AAV mediated exendin-4 gene transfer to salivary glands to protect subjects from diabetes or obesity

ABSTRACT

The invention relates to a gene transfer-based method to protect a subject from diabetes or obesity. The method comprises administering to a salivary gland of the subject an AAV virion comprising an AAV vector that encodes an exendin-4 protein. Also provided are exendin-4 proteins and nucleic acid molecules that encode such exendin-4 proteins. Also provided are AAV vectors and AAV virions that encode an exendin-4 protein. One embodiment is an exendin-4 protein that is a fusion protein comprising an NGF secretory segment joined to the amino terminus of an exendin-4 protein domain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/368,940, filed Dec. 5, 2016; which is a divisional of U.S. patentapplication Ser. No. 14/112,790, filed Mar. 20, 2014, issued as U.S.Pat. No. 9,511,103; which is a national stage application under 35U.S.C. 371 of PCT Application No. PCT/US2012/034268 having aninternational filing date of 19 Apr. 2012, which designated the UnitedStates; which PCT application claimed the benefit of U.S. ProvisionalApplication No. 61/477,523 filed Apr. 20, 2011; the entire disclosure ofeach of which is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named“Seq_Listing_6137NIDCR-13-PUS_Sequence_Listing_ST25.txt”, having a sizein bytes of 18 KB, and created on Oct. 7, 2013. The informationcontained in this electronic file is hereby incorporated by reference inits entirety pursuant to 37 CFR § 1.52(e)(5).

FIELD

The present invention relates to the use of gene therapy to protect asubject from diabetes or obesity. More specifically, the presentinvention relates to adeno-associated virus vectors and virions thatencode an exendin-4 protein and to their use to deliver nucleic acidmolecules encoding an exendin-4 protein to the salivary glands in orderto protect a subject from diabetes or obesity.

BACKGROUND

Glucagon-like peptide 1 (GLP-1), a hormone mainly produced in anutrient-dependent manner by gastrointestinal endocrine L cells (see,for example, Parker et al., 2010, Expert Rev Mol Med 12:e1), enhancesglucose-dependent insulin secretion and inhibits food intake, gastricemptying, and glucagon release, thus promoting the maintenance of normalglucose homeostasis (see, for example, Lauffer et al., 2009, Diabetes58, 1058-1066; Gribble, 2008, Diabet Med 25, 889-894). A small, butsignificant, defect in mixed meal and oral glucose load stimulated GLP-1secretion has been observed in Type 2 Diabetes (T2DM) (see for example,Mannucci et al., 2000, Diabet Med 17, 713-719; Vilsboll et al., 2001,Diabetes 50, 609-613). In Type 2 diabetic patients, chronicadministration of native GLP-1, via continuous infusion or repeatedsubcutaneous injection, reduces fasting and postprandial blood glucoseand decreases glycosylated hemoglobin (HbA1c) in association with amodest, but significant weight loss (see, for example, Zander et al.,2002, Lancet 359, 824-830; Meneilly et al., 2003, Diabetes Care 262835-2841). The short half-life of native GLP-1, due to rapidinactivation mainly catalyzed by dipeptidyl-peptidase-4 (DDP-4), hasengendered interest in the development of more stable longer-actingGLP-1 receptor agonists to be used as hypoglycemic drugs for thetreatment of T2DM. Exendin-4 (Ex-4), a peptide isolated from thesalivary secretion of the Gila monster, is a potent GLP-1 receptoragonist, which, because of its molecular structure, is considerably moreresistant than native GLP-1 to degradation by DPP-4 (see, for example,Neumiller, 2009, J Am Pharm Assoc 49 (suppl. 1, S16-S29). Exenatide (thesynthetic form of exendin-4, brand name BYETTA®) significantly improvesglycemic control and causes weight loss in type 2 diabetic patients(see, for example, Madsbad, 2009, Best Pract Res Clin Endocrinol Metab23, 463-477). Exenatide, which has been approved for the treatment toType 2 Diabetes, requires twice daily subcutaneous administration.

Gene therapy offers the possibility of more stable long-term expressionfor the treatment of many chronic diseases, including T2DM (Srivastava,2008, J Cell Biochem 105, 17-24). Recently, adenoviral and plasmid-basedvectors have been used to express GLP-1 receptor agonists in severaltissues, but have not resulted in long-term effects, as a result ofeither low or transient expression (see, for example, Voutetakis et al.,2010, Endocrinology 151, 4566-4572; Kumar et al., 2007, Gene Ther 14,162-172; Liu et al., 2010, Biochem Biophys Res Commun 403, 172-177;Samson et al., 2008, Mol Ther 16, 1805-1812 (erratum in Mol Ther 17,1831); Lee et al., 2008, J Gene Med 10, 260-268; Choi et al., 2005, MolTher 12, 885-891; Lee et al., 2007, Diabetes 56, 1671-1679). Whileeffective in animal models, the inherent risk profile related tosystemic delivery of vectors supported site-specific gene therapeuticapproaches as an appealing alternative.

Recently, adeno-associated viruses (AAVs) have advanced to the forefrontof gene therapy, due to their ability to achieve long-term transgeneexpression in vivo and low immunogenicity (see, for example,Sumner-Jones et al., 2006 Gene Ther 13, 1703-1713; Stieger et al., 2006,Mol Ther 13, 967-975; Niemeyer et al., 2009, Blood 113, 797-806; Daya etal., 2008, Clin Microbiol Rev 21, 583-593). Several Phase I/II clinicaltrials support a good overall safety profile for AAV vectors and littleassociated toxicity in humans (see, for example, Mandel, 2010 Curr OpinMol Ther 12, 240-247; Bainbridge et al., 2008, N Engl J Med 358,2231-2239; Moss et al., 2004, Chest 125, 509-521; Diaz-Nido, 2010, CurrOpin Investig Drugs 11, 813-822; Simonelli et al., 2010, Mol Ther 18,643-650). Over 100 AAV isolates have been reported; biochemical andmolecular characterization suggests that some exhibit different tissuetropism, persistence, and transduction efficiency (see, for example,Kwon et al., 2008, Pharm Res 25, 489-499). Among AAVs, serotype 5 (AAV5)has demonstrated enhanced gene transfer activity in lung, eye and CNS aswell as rodent salivary glands (SG) (see, for example, Katano et al.,2006, Gene Ther 13, 594-601.

Salivary glands are recognized as a useful depot organ in gene therapy,having several important features of other endocrine glands, such ashigh protein production and ability to secrete proteins into thebloodstream (see, for example, Voutetakis et al., 2005, J Endocrinol185, 363-372). It has been previously reported that salivary glands areable to produce pharmacological levels of growth hormone and parathyroidhormone following transduction with recombinant viral vectors (see, forexample, He et al., 1998, Gene Ther 5, 537-541; Adriaansen et al., 2011,Hum Gene Ther 22, 84-92).

There still remains a need for an effective and safe composition toprotect subjects from diabetes or obesity.

SUMMARY

The disclosure provides a gene transfer-based method to protect asubject from diabetes or obesity. The disclosure provides a genetransfer-based method to protect a subject from diabetes. The methodcomprises administering to a salivary gland of a subject anadeno-associated virus (AAV) virion comprising an AAV vector thatencodes an exendin-4 protein. The disclosure also provides a genetransfer-based method to protect a subject from obesity. The methodcomprises administering to a salivary gland of a subject anadeno-associated virus (AAV) virion comprising an AAV vector thatencodes an exendin-4 protein. In one embodiment, the exendin-4 proteincomprises an exendin-4 fusion protein comprising a secretory segment,such as an NGF secretory segment, joined to the amino terminus of anexendin-4 protein domain. Also provided are methods to produce suchexendin-4 proteins, AAV vectors encoding such exendin-4 proteins, andAAV virions comprising such AAV vectors. Also provided are nucleic acidmolecules that encode exendin-4 proteins of the embodiments and usesthereof.

The disclosure provides an exendin-4 protein, wherein the exendin-4protein comprises an exendin-4 fusion protein comprising a NGF secretorysegment joined to the amino terminus of an exendin-4 protein domain.

The disclosure provides an AAV vector that encodes an exendin-4 proteincomprising an exendin-4 fusion protein comprising a secretory segmentjoined to the amino terminus of an exendin-4 protein domain. Thedisclosure also provides an AAV virion that comprises an AAV vector thatencodes an exendin-4 protein comprising an exendin-4 fusion proteincomprising a secretory segment joined to the amino terminus of anexendin-4 protein domain. Also provided are AAV vectors that encodeother exendin-4 proteins of the embodiments, and AAV virions thatcomprise such AAV vectors.

The disclosure provides a treatment for diabetes. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes anexendin-4 protein. Administration of such a treatment to a subjectprotects the subject from diabetes.

The disclosure provides a treatment for obesity. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes anexendin-4 protein. Administration of such a treatment to a subjectprotects the subject from obesity.

The disclosure provides a preventative for diabetes. Such a preventativecomprises an AAV virion comprising an AAV vector that encodes anexendin-4 protein. Administration of such a preventative to a subjectprotects the subject from diabetes.

The disclosure provides a preventative for obesity. Such a preventativecomprises an AAV virion comprising an AAV vector that encodes anexendin-4 protein. Administration of such a preventative to a subjectprotects the subject from obesity.

The disclosure provides a salivary gland cell transfected with an AAVvector that encodes an exendin-4 protein. The salivary gland cell can bethat of a subject that is diabetic or obese.

The disclosure provides an AAV virion comprising an AAV vector thatencodes an exendin-4 protein for the protection of a subject fromdiabetes or obesity. The disclosure provides for the use of an AAVvirion comprising an AAV vector that encodes an exendin-4 protein forthe manufacture of a medicament to protect a subject from diabetes orobesity.

The disclosure provides a gene transfer-based method to protect asubject from an incretin defect. The method comprises administering to asalivary gland of a subject an adeno-associated virus (AAV) virioncomprising an AAV vector that encodes a GLP-1 analog protein, whereinsuch administration protects the subject from a disease due to anincretin defect. The disclosure provides a gene transfer-based method toprotect a subject from diabetes. The method comprises administering to asalivary gland of a subject an adeno-associated virus (AAV) virioncomprising an AAV vector that encodes a GLP-1 analog protein. Thedisclosure also provides a gene transfer-based method to protect asubject from obesity. The method comprises administering to a salivarygland of a subject an adeno-associated virus (AAV) virion comprising anAAV vector that encodes a GLP-1 analog protein. In one embodiment, theGLP-1 analog protein comprises a GLP-1 analog fusion protein comprisinga secretory segment, such as an NGF secretory segment, joined to theamino terminus of a GLP-1 analog protein domain. Also provided aremethods to produce such GLP-1 analog proteins, AAV vectors encoding suchGLP-1 analog proteins, and AAV virions comprising such AAV vectors. Alsoprovided are nucleic acid molecules that encode GLP-1 analog proteins ofthe embodiments and uses thereof.

The disclosure provides a GLP-1 analog protein, wherein the GLP-1 analogprotein comprises a GLP-1 analog fusion protein comprising a NGFsecretory segment joined to the amino terminus of a GLP-1 analog proteindomain.

The disclosure provides an AAV vector that encodes a GLP-1 analogprotein comprising a GLP-1 analog fusion protein comprising a secretorysegment joined to the amino terminus of a GLP-1 analog protein domain.The disclosure also provides an AAV virion that comprises an AAV vectorthat encodes a GLP-1 analog protein comprising a GLP-1 analog fusionprotein comprising a secretory segment joined to the amino terminus of aGLP-1 analog protein domain. Also provided are AAV vectors that encodeother GLP-1 analog proteins of the embodiments, and AAV virions thatcomprise such AAV vectors.

The disclosure provides a treatment for an incretin defect. Such atreatment comprises an AAV virion comprising an AAV vector that encodesa GLP-1 analog protein. Administration of such a treatment to a subjectprotects the subject from a disease due to such incretin defect.

The disclosure provides a treatment for diabetes. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes a GLP-1analog protein. Administration of such a treatment to a subject protectsthe subject from diabetes.

The disclosure provides a treatment for obesity. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes a GLP-1analog protein. Administration of such a treatment to a subject protectsthe subject from obesity.

The disclosure provides a preventative for an incretin defect. Such apreventative comprises an AAV virion comprising an AAV vector thatencodes a GLP-1 analog protein. Administration of such a preventative toa subject protects the subject from a disease due to such incretindefect.

The disclosure provides a preventative for diabetes. Such a preventativecomprises an AAV virion comprising an AAV vector that encodes a GLP-1analog protein. Administration of such a preventative to a subjectprotects the subject from diabetes.

The disclosure provides a preventative for obesity. Such a preventativecomprises an AAV virion comprising an AAV vector that encodes a GLP-1analog protein Administration of such a preventative to a subjectprotects the subject from obesity.

The disclosure provides a salivary gland cell transfected with an AAVvector that encodes a GLP-1 analog protein. The salivary gland cell canbe that of a subject that has an incretin defect. The salivary glandcell can be that of a subject that is diabetic or obese.

The disclosure provides an AAV virion comprising an AAV vector thatencodes a GLP-1 analog protein for the protection of a subject from anincretin defect. The disclosure provides for the use of an AAV virioncomprising an AAV vector that encodes a GLP-1 analog protein for themanufacture of a medicament to protect a subject from an incretindefect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates exendin-4 serum levels in High Fat-Diet (HFD) mice(at 42 days) and Zucker fa/fa rats (at 30 and 60 days) after salivarygland administration of AAV virion AAV5-CMV-NGF-Ex4, also referred toherein as AAV5-NGF-Ex4 and AAV5-Ex4. Exendin-4 protein levels wereassayed by a specific Enzyme Immunoassay (EIA) kit. Exendin-4 wasexpressed as mean values (pmol/L) in a logarithmic scale±standard error(SE).

FIG. 2 provides an epifluorescence microscopic image of salivary glandsof AAV virion AAV5-Ex4 treated (FIG. 2A) or control HFD mice (FIG. 2B);barr=20 Salivary gland tissue sections, adequately removed andcollected, were incubated with a primary antibody against exendin-4(Phoenix Pharmaceuticals Inc.) for 24 hours at 4° C. at a final dilutionof 1:50. Subsequently, the sections were incubated with an Alexa Fluor488 secondary donkey anti-rabbit antibody at a final dilution of 1:333for 2 hours at room temperature. The immunoreaction products wereobserved under an epifluorescence Zeiss Axioskop microscope at ×40magnification.

FIGS. 3A and 3B demonstrate loss of weight gain in HFD mice and Zuckerfa/fa rats at specified times after salivary gland administration of AAVvirion AAV5-Ex4 compared to that of virion control. Specifically, FIG.3A demonstrates loss of weight gain of HFD mice following administrationof AAV virion AAV5-Ex4 compared to virion control. Each group (AAVvirion-treated or control) was composed of ten mice, and the graphsrepresent the average weight gain values (g)±standard error (SE). Weightgain is expressed as difference (g) between weight at study point andbaseline value. ¥=p<0.01. FIG. 3B demonstrates loss of weight gain inZucker fa/fa rats following administration of AAV virion AAV5-Ex4compared to virion control. Each group (AAV virion-treated or control)was composed of five rats, and the graphs represent the average weightgain values (g)±standard error (SE). Weight gain is expressed asdifference (g) between weight at study point and baseline value.*=p<0.05.

FIG. 4 demonstrates results of an intraperitoneal insulin tolerancetest. At day 41, each animal was fasted for 4 hours. At 0 minutes,insulin (Humulin R Regular, Lilly) was intraperitoneally injected at 1unit/kg and samples were taken at specified times thereafter. Each group(AAV virion-treated or control) was composed of ten HFD mice, and thegraphs represent the average glycemic values (mmol/L)±standard error(SE). *=p<0.05.

FIGS. 5A and 5B demonstrate daily amount of food consumption in HFD miceand Zucker fa/fa rats at specified times after salivary glandadministration of AAV virion AAV5-Ex4 compared to virion control.Specifically, FIG. 5A demonstrates daily amount of High Fat Dietconsumption in HFD mice. Each group (AAV virion-treated or control) wascomposed often mice, and the graphs represent the average foodconsumption values (g/day)±standard error (SE). FIG. 5B demonstratesdaily amount of food consumption in Zucker fa/fa rats. Each group (AAVvirion-treated or control) was composed of five rats and the graphsrepresent the average food consumption values (g/day)±standard error(SE). * p<0.05.

FIG. 6 demonstrates short-term food intake in Zucker fa/fa rats atspecified times 30 days after salivary gland administration of AAVvirion AAV5-Ex4 compared to virion control. Each group (AAVvirion-treated or control) was composed of five rats, and the graphsrepresent the average food consumption values (g)±standard error (SE).¥=p<0.01.

FIG. 7 is a schematic map of plasmid vector pAAV5-GFP beta actin (diPasquale et al., 2005, Mol Ther 11, 849-855). Plasmid vector pAAV5-GFPbeta actin is 7495 base pairs (bp). The L ITR spans nucleotides 2through 200 of pAAV5-GFP beta actin. The CMV promoter domain spansnucleotides 212-802 of pAAV5-GFP beta actin. To produce plasmid vectorpAAV5-NGF-Ex4, the fragment spanning from the NheI restriction enzymesite at nucleotide 805 to the SmaI restriction enzyme site at nucleotide1637 of pAAV5-GFP beta actin was replaced with the NheI to SmaI nucleicacid molecule encoding mouse NGF secretory segment joined to the aminoterminus of Gila monster (Heloderma suspectum) exendin-4 (SEQ ID NO:3)to form plasmid vector pAAV5-NGF-Ex4 (see FIG. 8). The LacZ stufferspans nucleotides 2685-4205 of pAAV5-GFP beta actin. The R ITR spansnucleotides 4841-4598 of pAAV5-GFP beta actin.

FIG. 8 is a schematic map of plasmid vector pAAV5-NGF-Ex4, produced asdescribed in the description of FIG. 7 and in the Examples. Plasmidvector pAAV5-NGF-Ex4 is 7166 base pairs (bp). The L ITR spansnucleotides 2 through 200. The CMV promoter domain spans nucleotides212-802. The location of the NheI-SmaI NGF-Ex4 expression cassette (SEQID NO:3) encoding fusion protein NGF-Ex4 (i.e., a NGF secretory segmentjoined to the amino terminus of an exendin-4 protein) described in FIG.7 and the Examples is indicated as are the locations of the start codon(nucleotide 818-820) and stop codon (nucleotide 1301-1303) of theencoding fusion protein. The GFP □-actin-Lacz staffer spans nucleotides1304-3876. The R ITR spans nucleotides 4512-3876.

FIG. 9 provides the nucleic acid sequence of the pAAV-NGF-Ex4 cassette(SEQ ID NO:1). This sequence corresponds to nucleotides 605 through 1504of the AAV5 NGF-Ex4 plasmid depicted in FIG. 8.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It should be understood that as used herein, the term “a” entity or “an”entity refers to one or more of that entity. For example, a nucleic acidmolecule refers to one or more nucleic acid molecules. As such, theterms “a”, “an”, “one or more” and “at least one” can be usedinterchangeably. Similarly the terms “comprising”, “including” and“having” can be used interchangeably.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

The disclosure provides a novel gene therapy to protect a subject fromdiabetes or obesity. The inventors have discovered that administrationof an adeno-associated virus (AAV) virion comprising an AAV vector thatencodes an exendin-4 protein to a salivary gland of a subject protectsthat subject from diabetes or obesity. For example, administration of anAAV virion comprising an AAV vector that encodes an exendin-4 protein ofthe embodiments to salivary glands leads to sustained, site-specificexpression of exendin-4, which is secreted into the bloodstream, leadingto an improved weight profile and improvements in glucose homeostasisand in other metabolic effects. This discovery is surprising becauseprotein sorting in the salivary gland is unpredictable; see, forexample, Voutetakis et al., 2008, Hum Gene Ther 19, 1401-1405, and Perezet al., 2010, Int J Biochem Cell Biol 42, 773-777, Epub 2010 Feb. 26.Thus, one skilled in the art could not predict whether an exendin-4protein of the embodiments would sort in such a manner as to protect asubject from diabetes or obesity if a nucleic acid molecule encodingsuch a protein were delivered to a salivary gland of the subject (i.e.,whether exendin-4 produced by the salivary glands would traffic throughthe cell via the endocrine pathway, resulting in circulating serumlevels of the protein), or if the exendin-4 protein would sort in such amanner as to not have an effect on the subject, in view of aninsufficient amount of exendin-4 protein being secreted into thebloodstream.

Proteins

As used herein, an exendin-4 protein is any protein that exhibitsactivity of a natural exendin-4, such as the ability to bind to a GLP-1receptor and effect an agonist response at that receptor. An exendin-4protein can also exhibit a longer half-life than natural GLP-1 andexhibit increased resistance to dipeptidyl peptidase 4 compared toGLP-1. An exendin-4 protein of the embodiments can have a wild-typeexendin-4 sequence (i.e., it has the same amino acid sequence as anatural exendin-4), can be a portion of a natural exendin-4, or can be amutant of a natural exendin-4, provided that such a portion or mutantretains the ability to effect an agonist response at the GLP-1 receptor.

In one embodiment, an exendin-4 protein comprises an entire naturalexendin-4. In one embodiment, an exendin-4 protein is a portion of anatural exendin-4, wherein such portion retains the ability to effect anagonist response at the GLP-1 receptor and exhibit a longer half-lifethan natural GLP-1. In one embodiment, an exendin-4 protein is a mutantof a natural exendin-4, wherein such mutant retains the ability toeffect an agonist response at the GLP-1 receptor and exhibit a longerhalf-life than natural GLP-1. In one embodiment, an exendin-4 protein isa portion of a mutant of a natural exendin-4, wherein such exendin-4protein retains the ability to effect an agonist response at the GLP-1receptor and exhibit a longer half-life than natural GLP-1.

Methods to produce portions and mutants, such as conservative mutants,are known to those skilled in the art. Assays to determine bindingbetween an exendin-4 protein and a GLP-1 receptor and to determine theability of exendin-4 to effect an agonist response at the GLP-1 receptorare known to those skilled in the art, as are methods to measure thehalf-life of a protein; see, for example, Doyle et al., 2003, Regul Pept114, 153-158, and Examples herein. Thus, one skilled in the art canproduce portions or mutants of exendin-4 that bind to a GLP-1 receptor,effect an agonist response at a GLP-1 receptor, and/or exhibit a longerhalf-life than a natural GLP-1 protein without undue experimentation.

An exendin-4 protein of the embodiments can be derived from any speciesthat expresses functional exendin-4. In one embodiment, an exendin-4protein is derived from a species for which the protein is notimmunogenic in the subject being protected from diabetes or obesity.

One embodiment of the disclosure is an exendin-4 protein that comprisesa secretory segment (i.e., a secretory sequence) joined to the aminoterminus of an exendin-4 protein domain. Such an exendin-4 protein ofthe embodiments is referred to as an exendin-4 fusion protein. Theexendin-4 protein domain, or exendin-4 domain, in such an embodiment isthe portion of the fusion protein that has an exendin-4 amino acidsequence. As used herein, join refers to combine by attachment usinggenetic engineering techniques. In such an embodiment, an exendin-4protein can be joined directly to a secretory segment, or an exendin-4protein can be linked to the secretory segment by a linker of one ormore amino acids. A secretory segment enables an expressed exendin-4protein to be secreted from the cell that produces the protein. Asuitable secretory segment is a secretory segment that directs endocrinesecretion of an exendin-4 protein in the salivary glands. The inventorshave found, surprisingly, that a nerve growth factor (NGF) secretorysegment is particularly effective at directing endocrine secretion of anexendin-4 protein in the salivary glands. For example, endocrinesecretion is more effective with a NGF secretory segment than with aFactor IX secretory segment. In one embodiment, the secretory segment ismodified so as to be susceptible to cleavage from the exendin-4 proteindomain by a furin protease. One embodiment is a NGF secretory segmentthat is cleavable from the exendin-4 protein domain by a furin protease.One example is a secretory segment having SEQ ID NO:10. In oneembodiment, an exendin-4 protein is a fusion protein comprising a NGFsecretory segment joined to the amino terminus of an exendin-4 proteindomain.

Another embodiment of the disclosure is an exendin-4 protein joined to afusion segment; such a protein is another type of exendin-4 fusionprotein. Such a protein has an exendin-4 protein domain and a fusionsegment, and can also include a secretory segment. A fusion segment isan amino acid segment of any size that can enhance the properties of anexendin-4 protein; a fusion segment of the embodiments can, for example,increase the stability of an exendin-4 protein, add flexibility orenable multimerization, e.g., dimerization. Examples of fusion segmentsinclude, without being limited to, an immunoglobulin fusion segment, analbumin fusion segment, and any other fusion segment that increases thebiological half-life of the protein, provides flexibility to theprotein, and/or enables multimerization. It is within the scope of thedisclosure to use one or more fusion segments. Fusion segments can bejoined to the amino terminus and/or carboxyl terminus of an exendin-4protein of the embodiments. As used herein, join refers to combine byattachment using genetic engineering techniques. In such an embodiment,an exendin-4 protein can be joined directly to a fusion segment, or anexendin-4 protein can be linked to the fusion segment by a linker of oneor more amino acids.

One embodiment is an exendin-4 fusion protein that comprises anexendin-4 protein domain and an immunoglobulin fusion segment. Such anexendin-4 fusion protein can optionally also include a secretorysegment. Examples of immunoglobulin fusion segments include one or moreconstant regions of an immunoglobulin, such as one or more constantregions of gamma, mu, alpha, delta or epsilon Ig heavy chains or ofkappa or lambda Ig light chains. In one embodiment, an immunoglobulinfusion segment is at least one constant region of a gamma heavy chain.In one embodiment, an immunoglobulin fusion segment comprises the Fcregion of an immunoglobulin. The Fc region of an IgG, IgA, or IgDantibody comprises the hinge and second and third constant regions(i.e., CH2 and CH3) of the respective antibody. The Fc region of an IgMantibody comprises the hinge and second, third and fourth constantregions (CH2, CH3 and CH4) of the respective antibody. In oneembodiment, the immunoglobulin fusion segment comprises the Fc region ofan IgG, such as IgG1. In one embodiment, the immunoglobulin fusionsegment is an IgG C□1 (IgG C-gamma-1) segment. In one embodiment, theimmunoglobulin fusion segment is a human IgG C□1 segment.

One embodiment of the disclosure is an exendin-4 protein comprisingamino acid sequence SEQ ID NO:8. SEQ ID NO:8 is a 40-amino acid sequenceof Gila monster (Heloderma suspectum) exendin-4. One embodiment is anexendin-4 protein that is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%identical to amino acid sequence SEQ ID NO:8. In one embodiment, anexendin-4 protein is at least 60% identical to amino acid sequence SEQID NO:8. In one embodiment, an exendin-4 protein is at least 65%identical to amino acid sequence SEQ ID NO:8. In one embodiment, anexendin-4 protein is at least 70% identical to amino acid sequence SEQID NO:8. In one embodiment, an exendin-4 protein is at least 75%identical to amino acid sequence SEQ ID NO:8. In one embodiment, anexendin-4 protein is at least 80% identical to amino acid sequence SEQID NO:8. In one embodiment, an exendin-4 protein is at least 85%identical to amino acid sequence SEQ ID NO:8. In one embodiment, anexendin-4 protein is at least 90% identical to amino acid sequence SEQID NO:8. In one embodiment, an exendin-4 protein is at least 95%identical to amino acid sequence SEQ ID NO:8. In each of theseembodiments, the respective exendin-4 protein retains the ability toeffect an agonist response at a GLP-1 receptor. In one embodiment, suchan exendin-4 protein also comprises a fusion segment.

One embodiment is an exendin-4 fusion protein comprising a secretorysegment joined to the amino terminus of an exendin-4 domain, wherein theexendin-4 domain of the fusion protein is at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, or atleast 95% identical to amino acid sequence SEQ ID NO:8. One embodimentis an exendin-4 fusion protein, wherein the exendin-4 domain of thefusion protein is at least 60% identical to amino acid sequence SEQ IDNO:8. One embodiment is an exendin-4 fusion protein, wherein theexendin-4 domain of the fusion protein is at least 65% identical toamino acid sequence SEQ ID NO:8. One embodiment is an exendin-4 fusionprotein, wherein the exendin-4 domain of the fusion protein is at least70% identical to amino acid sequence SEQ ID NO:8. One embodiment is anexendin-4 fusion protein, wherein the exendin-4 domain of the fusionprotein is at least 75% identical to amino acid sequence SEQ ID NO:8.One embodiment is an exendin-4 fusion protein, wherein the exendin-4domain of the fusion protein is at least 80% identical to amino acidsequence SEQ ID NO:8. One embodiment is an exendin-4 fusion protein,wherein the exendin-4 domain of the fusion protein is at least 85%identical to amino acid sequence SEQ ID NO:8. One embodiment is anexendin-4 fusion protein, wherein the exendin-4 domain of the fusionprotein is at least 90% identical to amino acid sequence SEQ ID NO:8.One embodiment is an exendin-4 fusion protein, wherein the exendin-4domain of the fusion protein is at least 95% identical to amino acidsequence SEQ ID NO:8. One embodiment is an exendin-4 fusion proteincomprising an exendin-4 domain having amino acid SEQ ID NO:8. In each ofthese embodiments, the respective exendin-4 protein retains the abilityto effect an agonist response at a GLP-1 receptor. In one embodiment,such an exendin-4 protein also comprises a fusion segment.

One embodiment is an exendin-4 fusion protein comprising a secretorysegment joined to the amino terminus of an exendin-4 domain, wherein theexendin-4 fusion protein is at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%identical to amino acid sequence SEQ ID NO:2. Amino acid sequence SEQ IDNO:2 represents the sequence of a fusion protein of a mouse NGFsecretory segment (SEQ ID NO:10) joined to the amino acid terminus ofamino acid sequence SEQ ID NO:8. One embodiment is an exendin-4 fusionprotein that is at least 60% identical to amino acid sequence SEQ IDNO:2. One embodiment is an exendin-4 fusion protein that is at least 65%identical to amino acid sequence SEQ ID NO:2. One embodiment is anexendin-4 fusion protein that is at least 70% identical to amino acidsequence SEQ ID NO:2. One embodiment is an exendin-4 fusion protein thatis at least 75% identical to amino acid sequence SEQ ID NO:2. Oneembodiment is an exendin-4 fusion protein that is at least 80% identicalto amino acid sequence SEQ ID NO:2. One embodiment is an exendin-4fusion protein that is at least 85% identical to amino acid sequence SEQID NO:2. One embodiment is an exendin-4 fusion protein that is at least90% identical to amino acid sequence SEQ ID NO:2. One embodiment is anexendin-4 fusion protein that is at least 95% identical to amino acidsequence SEQ ID NO:2. One embodiment is an exendin-4 fusion proteincomprising amino acid SEQ ID NO:2. In each of these embodiments, therespective exendin-4 protein retains the ability to effect an agonistresponse at a GLP-1 receptor. In one embodiment, such an exendin-4protein also comprises a fusion segment.

One embodiment is an exendin-4 protein having an amino acid sequenceselected from the group consisting of amino acid sequence SEQ ID NO:2and SEQ ID NO:8. One embodiment is an exendin-4 protein having aminoacid sequence SEQ ID NO:2. One embodiment is an exendin-4 protein havingamino acid sequence SEQ ID NO:8.

In one embodiment, an exendin-4 protein is exendin-1; i.e., theexendin-4 protein has an amino acid sequence representative ofexendin-1. In one embodiment, an exendin-4 protein is not a gilatide,wherein a gilatide is a nine amino acid sequence as described in US Pub.No. 2004/0092432, published May 13, 2004.

The disclosure provides GLP-1 analog proteins that are encoded by AAVvectors of the embodiments. As used herein, a GLP-1 analog protein is aprotein, e.g., a peptide or larger protein, that binds to and effects anagonist response at a GLP-1 receptor. Examples of GLP-1 analog proteinsinclude, but are not limited to, exendin-4, exendin-1, lixisenatide,liraglutide, albiglutide, taspoglutide, dulaglutide, and semaglutide.Additional examples of GLP-1 analogs include those listed in PCTInternational Publication No. WO 03/011892, published Feb. 13, 2003, andHribal et al., 2011, Clin Invest 1, 327-343, both of which referencesare incorporated herein in their entireties. Additional non-limitingexamples are provided in Appendix A.

A GLP-1 analog protein can be a full-length protein, or a portion ormutant thereof. The embodiments include a GLP-1 analog fusion protein.In one embodiment, a GLP-1 analog fusion protein comprises a secretorysegment joined to the amino terminus of a GLP-1 analog protein domain.In one embodiment, a GLP-1 analog fusion protein comprises a fusionsegment joined to either the amino terminus or carboxyl terminus of aGLP-1 analog protein domain. One embodiment is a GLP-1 analog fusionprotein comprising both a secretory segment and a fusion segment.

Nucleic Acids

The disclosure provides nucleic acid molecules that encode an exendin-4protein of the embodiments. One embodiment is a nucleic acid moleculethat encodes an exendin-4 protein that is not a fusion protein. Oneembodiment is a nucleic acid molecule that encodes an exendin-4 fusionprotein comprising a secretory segment joined to the amino terminus ofan exendin-4 protein domain. One embodiment is a nucleic acid moleculethat encodes an exendin-4 fusion protein that comprises a fusion segmentjoined to an exendin-4 protein domain; such a fusion protein can alsocomprise a secretory segment joined to the amino terminus of theexendin-4 protein domain.

In one embodiment, a nucleic acid molecule encodes an exendin-4 proteincomprising amino acid sequence SEQ ID NO:8. One embodiment is a nucleicacid molecule that encodes an exendin-4 protein that is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% identical to amino acid sequence SEQ ID NO:8.In one embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 60% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 65% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 70% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 75% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 80% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 85% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, a nucleic acid molecule encodes an exendin-4 proteinthat is at least 90% identical to amino acid sequence SEQ ID NO:8. Inone embodiment, an exendin-4 protein is at least 95% identical to aminoacid sequence SEQ ID NO:8. In each of these embodiments, the exendin-4protein encoded by the respective nucleic acid molecule retains theability to effect an agonist response at a GLP-1 receptor. In oneembodiment, such a nucleic acid molecule also encodes a fusion segment.

In one embodiment, a nucleic acid molecule comprises nucleic acidsequence SEQ ID NO:7. Nucleic acid sequence SEQ ID NO:7 encodes aminoacid sequence SEQ ID NO:8. One embodiment is a nucleic acid moleculethat is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% identical to nucleic acid sequence SEQ ID NO:7. Oneembodiment is a nucleic acid molecule that is at least 70% identical tonucleic acid sequence SEQ ID NO:7. One embodiment is a nucleic acidmolecule that is at least 75% identical to nucleic acid sequence SEQ IDNO:7. One embodiment is a nucleic acid molecule that is at least 80%identical to nucleic acid sequence SEQ ID NO:7. One embodiment is anucleic acid molecule that is at least 85% identical to nucleic acidsequence SEQ ID NO:7. One embodiment is a nucleic acid molecule that isat least 90% identical to nucleic acid sequence SEQ ID NO:7. Oneembodiment is a nucleic acid molecule that is at least 95% identical tonucleic acid sequence SEQ ID NO:7. In each of these embodiments, theexendin-4 protein encoded by the respective nucleic acid moleculeretains the ability to effect an agonist response at a GLP-1 receptor.In one embodiment, such a nucleic acid molecule also encodes a fusionsegment.

One embodiment is a nucleic acid molecule that encodes an exendin-4fusion protein comprising a secretory segment joined to the aminoterminus of an exendin-4 fusion protein domain, wherein the encodedexendin-4 fusion protein domain is an amino acid sequence that is atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, or at least 95% identical to amino acidsequence SEQ ID NO:8. One embodiment is a nucleic acid molecule thatencodes an exendin-4 fusion protein, wherein the exendin-4 proteindomain is at least 60% identical to amino acid sequence SEQ ID NO:8. Oneembodiment is a nucleic acid molecule that encodes an exendin-4 fusionprotein, wherein the exendin-4 protein domain is at least 65% identicalto amino acid sequence SEQ ID NO:8. One embodiment is a nucleic acidmolecule that encodes an exendin-4 fusion protein, wherein the exendin-4protein domain is at least 70% identical to amino acid sequence SEQ IDNO:8. One embodiment is a nucleic acid molecule that encodes anexendin-4 fusion protein, wherein the exendin-4 protein domain is atleast 75% identical to amino acid sequence SEQ ID NO:8. One embodimentis a nucleic acid molecule that encodes an exendin-4 fusion protein,wherein the exendin-4 protein domain is at least 80% identical to aminoacid sequence SEQ ID NO:8. One embodiment is a nucleic acid moleculethat encodes an exendin-4 fusion protein, wherein the exendin-4 proteindomain is at least 85% identical to amino acid sequence SEQ ID NO:8. Oneembodiment is a nucleic acid molecule that encodes an exendin-4 fusionprotein, wherein the exendin-4 protein domain is at least 90% identicalto amino acid sequence SEQ ID NO:8. One embodiment is a nucleic acidmolecule that encodes an exendin-4 fusion protein, wherein the exendin-4protein domain is at least 95% identical to amino acid sequence SEQ IDNO:8. One embodiment is a nucleic acid molecule that encodes anexendin-4 fusion protein, wherein the exendin-4 protein domain comprisesamino acid SEQ ID NO:8. In each of these embodiments, the exendin-4fusion protein encoded by the respective nucleic acid molecule retainsthe ability to effect an agonist response at a GLP-1 receptor. In oneembodiment, such a nucleic acid molecule also encodes a fusion segment.

One embodiment is a nucleic acid molecule that encodes an exendin-4fusion protein comprising a secretory segment joined to the aminoterminus of an exendin-4 fusion protein domain, wherein the exendin-4protein domain is encoded by a nucleic acid molecule that is at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% identical to nucleic acid sequence SEQ ID NO:7. One embodiment is anucleic acid molecule that encodes an exendin-4 fusion protein, whereinthe exendin-4 protein domain is encoded by a nucleic acid molecule thatis at least 70% identical to nucleic acid sequence SEQ ID NO:7. Oneembodiment is a nucleic acid molecule that encodes an exendin-4 fusionprotein, wherein the exendin-4 protein domain is encoded by a nucleicacid molecule that is at least 75% identical to nucleic acid sequenceSEQ ID NO:7. One embodiment is a nucleic acid molecule that encodes anexendin-4 fusion protein, wherein the exendin-4 protein domain isencoded by a nucleic acid molecule that is at least 80% identical tonucleic acid sequence SEQ ID NO:7. One embodiment is a nucleic acidmolecule that encodes an exendin-4 fusion protein, wherein the exendin-4protein domain is encoded by a nucleic acid molecule that is at least85% identical to nucleic acid sequence SEQ ID NO:7. One embodiment is anucleic acid molecule that encodes an exendin-4 fusion protein, whereinthe exendin-4 protein domain is encoded by a nucleic acid molecule thatis at least 90% identical to nucleic acid sequence SEQ ID NO:7. Oneembodiment is a nucleic acid molecule that encodes an exendin-4 fusionprotein, wherein the exendin-4 protein domain is encoded by a nucleicacid molecule that is at least 95% identical to nucleic acid sequenceSEQ ID NO:7. One embodiment is a nucleic acid molecule that encodes anexendin-4 fusion protein, wherein the exendin-4 protein domain isencoded by a nucleic acid molecule comprising nucleic acid sequence SEQID NO:7. In each of these embodiments, the exendin-4 fusion proteinencoded by the respective nucleic acid molecule retains the ability toeffect an agonist response at a GLP-1 receptor. In one embodiment, sucha nucleic acid molecule also encodes a fusion segment.

One embodiment is a nucleic acid molecule that encodes an exendin-4fusion protein comprising a secretory segment joined to the aminoterminus of an exendin-4 fusion protein domain, wherein the exendin-4fusion protein comprises an amino acid sequence that is at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% identical to amino acid sequence SEQ ID NO:2.One embodiment is a nucleic acid molecule that encodes an exendin-4fusion protein that is at least 60% identical to amino acid sequence SEQID NO:2. One embodiment is a nucleic acid molecule that encodes anexendin-4 fusion protein that is at least 65% identical to amino acidsequence SEQ ID NO:2. One embodiment is a nucleic acid molecule thatencodes an exendin-4 fusion protein that is at least 70% identical toamino acid sequence SEQ ID NO:2. One embodiment is a nucleic acidmolecule that encodes an exendin-4 fusion protein that is at least 75%identical to amino acid sequence SEQ ID NO:2. One embodiment is anucleic acid molecule that encodes an exendin-4 fusion protein that isat least 80% identical to amino acid sequence SEQ ID NO:2. Oneembodiment is a nucleic acid molecule that encodes an exendin-4 fusionprotein that is at least 85% identical to amino acid sequence SEQ IDNO:2. One embodiment is a nucleic acid molecule that encodes anexendin-4 fusion protein that is at least 90% identical to amino acidsequence SEQ ID NO:2. One embodiment is a nucleic acid molecule thatencodes an exendin-4 fusion protein that is at least 95% identical toamino acid sequence SEQ ID NO:2. One embodiment is a nucleic acidmolecule that encodes an exendin-4 fusion protein comprising amino acidSEQ ID NO:2. In each of these embodiments, the exendin-4 fusion proteinencoded by the respective nucleic acid molecule retains the ability toeffect an agonist response at a GLP-1 receptor. In one embodiment, sucha nucleic acid molecule also encodes a fusion segment.

One embodiment is a nucleic acid molecule that encodes an exendin-4fusion protein comprising a secretory segment joined to the aminoterminus of an exendin-4 fusion protein domain, wherein the exendin-4fusion protein is encoded by a nucleic acid molecule that is at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% identical to nucleic acid sequence SEQ ID NO:5. Nucleic acidsequence SEQ ID NO:5 encodes amino acid sequence SEQ ID NO:6, which isidentical to amino acid sequence SEQ ID NO:2 and to amino acid sequenceSEQ ID NO:4. One embodiment is a nucleic acid molecule that is at least70% identical to nucleic acid sequence SEQ ID NO:5. One embodiment is anucleic acid molecule that is at least 75% identical to nucleic acidsequence SEQ ID NO:5. One embodiment is a nucleic acid molecule that isat least 80% identical to nucleic acid sequence SEQ ID NO:5. Oneembodiment is a nucleic acid molecule that is at least 85% identical tonucleic acid sequence SEQ ID NO:5. One embodiment is a nucleic acidmolecule that is at least 90% identical to nucleic acid sequence SEQ IDNO:5. One embodiment is a nucleic acid molecule that is at least 95%identical to nucleic acid sequence SEQ ID NO:5. One embodiment is anucleic acid molecule that comprises nucleic acid sequence SEQ ID NO:5.In each of these embodiments, the exendin-4 fusion protein encoded bythe respective nucleic acid molecule retains the ability to effect anagonist response at a GLP-1 receptor. In one embodiment, such a nucleicacid molecule also encodes a fusion segment.

One embodiment is a nucleic acid molecule encoding an exendin-4 proteinhaving an amino acid sequence selected from the group consisting ofamino acid sequence SEQ ID NO:2 and SEQ ID NO:8. One embodiment is anucleic acid molecule encoding an exendin-4 protein having amino acidsequence SEQ ID NO:2. One embodiment is a nucleic acid molecule encodingan exendin-4 protein having amino acid sequence SEQ ID NO:8.

One embodiment is a nucleic acid molecule having a nucleic acid sequenceselected from the group consisting of nucleic acid sequence SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. One embodiment is a nucleicacid molecule having nucleic acid sequence SEQ ID NO:1. One embodimentis a nucleic acid molecule having nucleic acid sequence SEQ ID NO:3. Oneembodiment is a nucleic acid molecule having nucleic acid sequence SEQID NO:5. One embodiment is a nucleic acid molecule having nucleic acidsequence SEQ ID NO:7.

The disclosure provides a nucleic acid molecule that encodes any GLP-1analog protein of the embodiments. One embodiment is a nucleic acidmolecule that encodes a GLP-1 analog protein that is not a fusionprotein. One embodiment is a nucleic acid molecule that encodes a GLP-1analog fusion protein comprising a secretory segment joined to the aminoterminus of an exendin-4 protein domain. One embodiment is a nucleicacid molecule that encodes a GLP-1 analog fusion protein that comprisesa fusion segment joined to a GLP-1 analog protein domain; such a fusionprotein can also comprise a secretory segment joined to the aminoterminus of the GLP-1 analog protein domain.

Vectors and Virions

Adeno-associated virus (AAV) is a unique, non-pathogenic member of theParvoviridae family of small, non-enveloped, single-stranded DNA animalviruses. AAV require helper virus (e.g., adenovirus) for replicationand, thus, do not replicate upon administration to a subject. AAV caninfect a relatively wide range of cell types and stimulate only a mildimmune response, particularly as compared to a number of other viruses,such as adenovirus. Over 100 AAV isolates have been reported.Biochemical and molecular characterization of many suggests that someexhibit different tissue tropism, persistence, and transductionefficiency (see, for example, Kwon et al., ibid.). Examples of AAVinclude AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, and AAV12, which appear to be of simian or human origin. AAV havealso been found in other animals, including birds (e.g., avian AAV, orAAAV), bovines (e.g., bovine AAV, or BAAV), canines, equines, ovines,and porcines.

Vectors and virions based upon AAV have advanced to the forefront ofgene therapy, due to their ability to achieve long-term transgeneexpression in vivo and low immunogenicity (see, for example, Halbert etal., 2000, J Virol 74, 1524-1532; Sumner-Jones et al., ibid.; Stieger etal., ibid.; Niemeyer et al., ibid.). AAV virions have hitherto not beenassociated with any malignant disease. Furthermore, all viral proteingenes can be deleted from AAV vectors and AAV virions contributing totheir safety profile (see, for example, Daya et al., ibid.). SeveralPhase I/II clinical trials support a good overall safety profile for AAVvirions and little associated toxicity in humans (see, for example, Mosset al., ibid.; Mandel et al., ibid., Diaz-Nido et al., ibid., Simonelliet al., ibid; Bainbridge et al., ibid.).

An AAV vector is a recombinant nucleic acid molecule in which at least aportion of the AAV genome is replaced by a heterologous nucleic acidmolecule. It is possible to replace about 4.7 kilobases (kb) of AAVgenome DNA, e.g., by removing the viral replication and capsid genes.Often the heterologous nucleic acid molecule is simply flanked by AAVinverted terminal repeats (ITRs) on each terminus. The ITRs serve asorigins of replication and contain cis acting elements required forrescue, integration, excision from cloning vectors, and packaging. Suchvectors typically also include a promoter operatively linked to theheterologous nucleic acid molecule to control expression.

An AAV vector can be packaged into an AAV capsid in vitro with theassistance of a helper virus or helper functions expressed in cells toyield an AAV virion. The serotype and cell tropism of an AAV virion areconferred by the nature of the viral capsid proteins.

AAV vectors and AAV virions have been shown to transduce cellsefficiently, including both dividing and non-dividing cells (see, forexample, Lai et al., 2002, DNA Cell Biol 21, 895-913). Among AAVs,serotype 5 (AAV5) has demonstrated enhanced gene transfer activity inlung, eye and central nervous system (CNS) as well as rodent salivaryglands (see, for example, Katano et al., ibid.). AAV vectors and virionshave been shown to be safe and to lead to long term in vivo persistenceand expression in a variety of cell types.

As used herein, an AAV vector that encodes an exendin-4 protein is anucleic acid molecule that comprises a nucleic acid molecule thatencodes an exendin-4 protein of the embodiments, an ITR joined to 5′terminus of the exendin-4 nucleic acid molecule, and an ITR joined tothe 3′ terminus of the exendin-4 nucleic acid molecule. Examples of ITRsinclude, but are not limited, to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAAV, BAAV, and other AAV ITRsknown to those skilled in the art. In one embodiment, an AAV ITR isselected from an AAV2 ITR, an AAV5 ITR, an AAV6 ITR, and a BAAV ITR. Inone embodiment, an AAV ITR is an AAV2 ITR. In one embodiment, an AAV ITRis an AAV5 ITR. In one embodiment, an AAV ITR is an AAV6 ITR. In oneembodiment, an AAV ITR is a BAAV ITR.

An AAV vector of the embodiments can also include other sequences, suchas expression control sequences. Examples of expression controlsequences include, but are not limited to, a promoter, an enhancer, arepressor, a ribosome binding site, an RNA splice site, apolyadenylation site, a transcriptional terminator sequence, and amicroRNA binding site. Examples of promoters include, but are notlimited to, an AAV promoter, such as a p5, p19 or p40 promoters, anadenovirus promoter, such as an adenoviral major later promoter, acytomegalovirus (CMV) promoter, a papilloma virus promoter, a polyomavirus promoter, a respiratory syncytial virus (RSV) promoter, a sarcomavirus promoter, an SV40 promoter other viral promoters, an actinpromoter, an amylase promoter, an immunoglobulin promoter, a kallikreinpromoter, a metallothionein promoter, a heat shock promoter, anendogenous promoter, a promoter regulated by rapamycin or other smallmolecules, other cellular promoters, and other promoters known to thoseskilled in the art. In one embodiment, the promoter is an AAV promoter.In one embodiment, the promoter is a CMV promoter. Selection ofexpression control sequences to include can be accomplished by oneskilled in the art.

The disclosure provides AAV vectors of different serotypes (asdetermined by the serotype of the ITRs within such vector) that encodean exendin-4 protein of the embodiments. Such an AAV vector can beselected from an AAV1 vector, an AAV2 vector, an AAV3 vector, an AAV4vector, an AAV5 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector,an AAV9 vector, an AAV10 vector, an AAV11 vector, an AAV12 vector, anAAAV vector, and a BAAV vector, and other AAV vectors known to thoseskilled in the art. wherein any of such vectors encode an exendin-4protein of the embodiments. One embodiment is an AAV2 vector, an AAV5vector, an AAV6 vector or a BAAV vector, wherein the respective vectorencodes an exendin-4 protein of the embodiments. One embodiment is anAAV2 vector that encodes an exendin-4 protein of the embodiments. Oneembodiment is an AAV5 vector that encodes an exendin-4 protein of theembodiments. One embodiment is an AAV6 vector that encodes an exendin-4protein of the embodiments. One embodiment is a BAAV vector that encodesan exendin-4 protein of the embodiments.

One embodiment is an AAV vector that comprises AAV ITRs and a CMVpromoter operatively linked to a nucleic acid molecule encoding anexendin-4 protein of the embodiments. One embodiment is an AAV vectorthat comprises AAV ITRs and a CMV promoter operatively linked to anucleic acid molecule encoding an exendin-4 fusion protein of theembodiments. One embodiment is an AAV5 vector that comprises AAV5 ITRsand a CMV promoter operatively linked to a nucleic acid moleculeencoding an exendin-4 protein of the embodiments. One embodiment is anAAV5 vector that comprises AAV5 ITRs and a CMV promoter operativelylinked to a nucleic acid molecule encoding an exendin-4 fusion proteinof the embodiments. One embodiment is an AAV5 vector that comprises AAV5ITRs and a CMV promoter operatively linked to a nucleic acid moleculeencoding a fusion protein comprising an NGF secretory segment joined toan exendin-4 fusion protein domain of the embodiments.

The disclosure provides plasmid vectors that encode an exendin-4 proteinof the embodiments. Such plasmid vectors also include control regions,such as AAV ITRs, a promoter operatively linked to the nucleic acidmolecule encoding the exendin-4 protein, one or more splice sites, apolyadenylation site, and a transcription termination site. Such plasmidvectors also typically include a number of restriction enzyme sites aswell as a nucleic acid molecule that encodes drug resistance. An exampleof a plasmid vector is pAAV5-NGF-Ex4, a schematic of which is shown inFIG. 8.

One embodiment is an AAV vector comprising a nucleic acid moleculeencoding an exendin-4 protein having an amino acid sequence selectedfrom the group consisting of amino acid sequence SEQ ID NO:2 and SEQ IDNO:8. One embodiment is an AAV vector comprising a nucleic acid moleculeencoding an exendin-4 protein having amino acid sequence SEQ ID NO:2.One embodiment is an AAV vector comprising a nucleic acid moleculeencoding an exendin-4 protein having amino acid sequence SEQ ID NO:8.

One embodiment is an AAV vector comprising a nucleic acid moleculehaving a nucleic acid sequence selected from the group consisting ofnucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ IDNO:7. One embodiment is an AAV vector comprising a nucleic acid moleculehaving nucleic acid sequence SEQ ID NO:1. One embodiment is an AAVvector comprising a nucleic acid molecule having nucleic acid sequenceSEQ ID NO:3. One embodiment is an AAV vector comprising a nucleic acidmolecule having nucleic acid sequence SEQ ID NO:5. One embodiment is anAAV vector comprising a nucleic acid molecule having nucleic acidsequence SEQ ID NO:7.

One embodiment is the AAV vector depicted in FIG. 8.

The disclosure provides an AAV virion. An AAV virion is an AAV vectorencoding an exendin-4 protein of the embodiments encapsidated in an AAVcapsid. Examples of AAV capsids include AAV1 capsids, AAV2 capsids, AAV3capsids, AAV4 capsids, AAV5 capsids, AAV6 capsids, AAV7 capsids, AAV8capsids, AAV9 capsids, AAV10 capsids, AAV11 capsids, AAV12 capsids, AAAVcapsids, BAAV capsids, and capsids from other AAV serotypes known tothose skilled in the art. In one embodiment, the capsid is a chimericcapsid, i.e., a capsid comprising VP proteins from more than oneserotype. As used herein, the serotype of an AAV virion of theembodiments is the serotype conferred by the VP capsid proteins. Forexample, an AAV2 virion is a virion comprising AAV2 VP1, VP2 and VP3proteins.

One embodiment of the disclosure is an AAV virion comprising an AAVvector that encodes an exendin-4 protein of the embodiments. Such an AAVvirion can be selected from an AAV1 virion, an AAV2 virion, an AAV3virion, an AAV4 virion, an AAV5 virion, an AAV6 virion, an AAV7 virion,an AAV8 virion, an AAV9 virion, an AAV10 virion, an AAV11 virion, anAAV12 virion, an AAAV virion, a BAAV virion, and AAV virions of otherAAV serotype known to those skilled in the art.

One embodiment of the disclosure is an AAV virion selected from an AAV2virion, an AAV5 virion, an AAV6 virion, and a BAAV virion, wherein theAAV vector within the virion encodes an exendin-4 protein of theembodiments. One embodiment is an AAV2 virion, wherein the AAV vectorwithin the virion encodes an exendin-4 protein of the embodiments. Oneembodiment is an AAV5 virion, wherein the AAV vector within the virionencodes an exendin-4 protein of the embodiments. One embodiment is anAAV6 virion, wherein the AAV vector within the virion encodes anexendin-4 protein of the embodiments. One embodiment is a BAAV virion,wherein the AAV vector within the virion encodes an exendin-4 protein ofthe embodiments.

One embodiment is an AAV virion comprising an AAV vector comprising anucleic acid molecule encoding an exendin-4 protein having an amino acidsequence selected from the group consisting of amino acid sequence SEQID NO:2 and SEQ ID NO:8. One embodiment is an AAV virion comprising anAAV vector comprising a nucleic acid molecule encoding an exendin-4protein having amino acid sequence SEQ ID NO:2. One embodiment is an AAVvirion comprising an AAV vector comprising a nucleic acid moleculeencoding an exendin-4 protein having amino acid sequence SEQ ID NO:8.

One embodiment is an AAV virion comprising an AAV vector comprising anucleic acid molecule having a nucleic acid sequence selected from thegroup consisting of nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, and SEQ ID NO:7. One embodiment is an AAV virion comprising anAAV vector comprising a nucleic acid molecule having nucleic acidsequence SEQ ID NO:1. One embodiment is an AAV virion comprising an AAVvector comprising a nucleic acid molecule having nucleic acid sequenceSEQ ID NO:3. One embodiment is an AAV virion comprising an AAV vectorcomprising a nucleic acid molecule having nucleic acid sequence SEQ IDNO:5. One embodiment is an AAV virion comprising an AAV vectorcomprising a nucleic acid molecule having nucleic acid sequence SEQ IDNO:7.

Methods useful for producing AAV vectors and AAV virions disclosedherein are known to those skilled in the art and are also exemplified inthe Examples. Briefly, an AAV vector of the embodiments can be producedusing recombinant DNA or RNA techniques to isolate nucleic acidsequences of interest and join them together as described herein, e.g.,by using techniques known to those skilled in the art, such asrestriction enzyme digestion, ligation, PCR amplification, and the like.Methods to produce an AAV virion of the embodiments typically include(a) introducing an AAV vector of the embodiments into a host, (b)introducing a helper vector into the host cell, wherein the helpervector comprises the viral functions missing from the AAV vector and (c)introducing a helper virus into the host cell. All functions for AAVvirion replication and packaging need to be present, to achievereplication and packaging of the AAV vector into AAV virions. In someinstances, at least one of the viral functions encoded by the helpervector can be expressed by the host cell. Introduction of the vectorsand helper virus can be carried out using standard techniques and occursimultaneously or sequentially. The host cells are then cultured toproduce AAV virions, which are then purified using standard techniques,such as CsCl gradients. Residual helper virus activity can beinactivated using known methods, such as heat inactivation. Such methodstypically result in high titers of highly purified AAV virions that areready for use. In some embodiments, an AAV vector of a specifiedserotype is packaged in a capsid of the same serotype. For example, anAAV2 vector can be packaged in an AAV2 capsid; an AAV5 vector can bepackaged in an AAV5 capsid; an AAV6 vector can be packaged in an AAV6capsid; or a BAAV vector can be packaged in a BAAV capsid. In otherembodiments, an AAV vector of a specified serotype is packaged in acapsid of a different serotype in order to modify the tropism of theresultant virion. Combinations of AAV vector serotypes and AAV capsidserotypes can be determined by those skilled in the art.

The disclosure provides an AAV virion that comprises an AAV vector thatencodes a GLP-1 analog protein of the embodiments, and its use forincretin-based therapy. As used herein, an incretin is agastrointestinal hormone that causes an increase in the amount ofinsulin released from beta cells of the islets of Langerhans aftereating. The disclosure also provides an AAV vector that encodes a GLP-1analog protein of the embodiments. Suitable AAV vectors and AAV virionsare described herein.

Compositions and Method of Use

The disclosure provides a composition comprising an AAV vector encodingan exendin-4 protein of the embodiments. The disclosure also provides acomposition comprising an AAV virion comprising an AAV vector encodingan exendin-4 protein of the embodiments. Such compositions can alsoinclude an aqueous solution, such as a physiologically compatiblebuffer. Examples of excipients include water, saline, Ringer's solution,and other aqueous physiologically balanced salt solutions. In someembodiments, excipients are added to, for example, maintain particlestability or to prevent aggregation. Examples of such excipientsinclude, but are not limited to, magnesium to maintain particlestability, pluronic acid to reduce sticking, mannitol to reduceaggregation, and the like, known to those skilled in the art.

A composition of the embodiments is conveniently formulated in a formsuitable for administration to a subject. Techniques to formulate suchcompositions are known to those skilled in the art. For example, an AAVvirion of the embodiments can be combined with saline or otherpharmaceutically acceptable solution; in some embodiments excipients arealso added. In another embodiment, a composition comprising an AAVvirion is dried, and a saline solution or other pharmaceuticallyacceptable solution can be added to the composition prior toadministration.

The disclosure provides a method to protect a subject from an indicationselected from the group consisting of diabetes and obesity. That is, thedisclosure provides a method to protect a subject from diabetes orobesity. In other words, the disclosure provides a method to protect asubject from diabetes, obesity or diabetes and obesity. As used herein,diabetes refers to diabetes mellitus, which is a group of relatedmetabolic diseases, including Type 1 diabetes, Type 2 diabetes,gestational diabetes, maturity onset diabetes of the young (MODY), andrelated diseases. As used herein, obesity refers to a medical conditionin which excess body fat has accumulated to the extent that it can havean adverse effect on a subject's health. Obesity can be measured by bodymass index (BMI), a measurement that compares height and weight.Typically a subject is considered to be obese if her/his BMI is greaterthan 30 kg/m².

Such a method includes the step of administering to a salivary gland ofthe subject an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments. As used herein, the ability of anAAV virion of the embodiments to protect a subject from diabetes orobesity refers to the ability of such AAV virion to prevent, treat, orameliorate symptoms of diabetes or obesity. In one embodiment,administration of an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments to the salivary glands of a subjectprevents one or more symptoms of diabetes. In one embodiment,administration of an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments to the salivary glands of a subjecttreats one or more symptoms of diabetes. In one embodiment,administration of an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments to the salivary glands of a subjectameliorates one or more symptoms of diabetes. In one embodiment,administration of an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments to the salivary glands of a subjectprevents one or more symptoms of obesity. In one embodiment,administration of an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments to the salivary glands of a subjecttreats one or more symptoms of obesity. In one embodiment,administration of an AAV virion comprising an AAV vector that encodes anexendin-4 protein of the embodiments to the salivary glands of a subjectameliorates one or more symptoms of obesity. In one embodiment, an AAVvirion of the embodiments prevents symptoms of diabetes or obesity fromoccurring in a subject, for example in a subject susceptible to diabetesor obesity. In one embodiment, an AAV virion of the embodiments preventssymptoms of diabetes or obesity from worsening. In one embodiment, anAAV virion of the embodiments reduces symptoms of diabetes or obesity ina subject. In one embodiment, an AAV virion of the embodiments enables asubject to recover from symptoms of diabetes or obesity. Protecting fromdiabetes can include controlling glycemic and extra-glycemic effects ofdiabetes. Protecting from diabetes can include reduced hyperglycemia.Protecting from diabetes can include decreased insulin resistance.Protecting from diabetes can include maintaining normal blood sugarlevels. Protecting from obesity can include increased energyexpenditure. Protecting from obesity can include an improved weightprofile. Protecting from obesity can include reducing a subject's BMI.Protecting from obesity can include maintaining a normal BMI in asubject, e.g., a BMI less than 30 kg/m². Protection from diabetes orobesity can include at least one of the following: increased circulationof biologically-active exendin-4 in the sera, reduced weight gain,reduced hyperglycemia, improvement in glucose homeostasis, reducedinsulin-induced glycemia, improvement in adipokine profile, reducedcirculating levels of leptin, reduced leptin expression in visceraladipose tissue, reduced circulating levels of HbA1c, reduced glycosuria,reduced insulin resistance, increased insulin sensitivity, increasedenergy expenditure, reduced food consumption, and reduced food intakefollowing fasting. Methods to measure such characteristics are known tothose skilled in the art and are described in the Examples.

One embodiment is protecting a subject from Type II diabetes. Oneembodiment is protecting a patient from Type I diabetes. One embodimentis protecting a subject from gestational diabetes. One embodiment isprotecting a subject from maturity onset diabetes of the young (MODY).One embodiment is protecting a subject from obesity. One embodiment isprotecting a subject from a monogenic form of obesity or diabetes (e.g.,Type 2 diabetes). One embodiment is protecting a subject from apolygenic form of obesity or diabetes (e.g., Type 2 diabetes).

As used herein, a subject is any animal that is susceptible to diabetesor obesity. Subjects include humans and other mammals, such as cats,dogs, horses, other companion animals, other zoo animals, lab animals(e.g., mice, rats), and livestock.

In accordance with the disclosure, an AAV virion of the embodiments isadministered to a salivary gland of a subject. Salivary glands havepotential as a target for gene therapy in some endocrine disorders,exhibiting several important features of endocrine glands, such ashighly efficient protein production and ability to secrete proteins intothe bloodstream primarily through a constitutive secretory pathway; see,for example, Voutetakis et al., 2005, ibid. However, protein sorting insalivary glands is unpredictable; see, for example, Voutetakis et al.,2008, Hum Gene Ther 19, 1401-1405, and Perez et al., 2010, Int J BiochemCell Biol 42, 773-777, Epub 2010 Feb. 26. As such, it is surprising thatthis administration route led to protection from diabetes or obesity.For example, it was surprising that salivary glands administered an AAVvirion encoding an exendin-4 fusion protein having a secretory segmentjoined to an exendin-4 protein domain are able to secrete the expressedprotein in order to effect protection from diabetes or obesity.Particularly surprising is that salivary glands administered an AAVvirion encoding an exendin-4 fusion protein having a NGF secretorysegment joined to an exendin-4 protein domain are able to secrete theexpressed protein in order to effect protection from diabetes orobesity.

In one embodiment an AAV virion of the embodiments is administered to asalivary gland of a subject. Such an AAV virion can be selected from anAAV1 virion, an AAV2 virion, an AAV3 virion, an AAV4 virion, an AAV5virion, an AAV6 virion, an AAV7 virion, an AAV8 virion, an AAV9 virion,an AAV10 virion, an AAV11 virion, an AAV12 virion, an AAAV virion, and aBAAV virion, and other AAV virions known to those skilled in the art,wherein any of such virions comprise an AAV vector that encodes anexendin-4 protein of the embodiments.

In one embodiment an AAV virion selected from an AAV2 virion, an AAV5virion, an AAV6 virion, and a BAAV virion, wherein the AAV virioncomprises an AAV vector that encodes an exendin-4 protein of theembodiments, is administered to a salivary gland. In one embodiment anAAV2 virion of the embodiments is administered to a salivary gland. Inone embodiment an AAV5 virion of the embodiments is administered to asalivary gland. In one embodiment an AAV6 virion of the embodiments isadministered to a salivary gland. In one embodiment an BAAV virion ofthe embodiments is administered to a salivary gland. Such administrationcan occur, for example, by cannulation, e.g., retrograde cannulation.

The disclosure also provides ex vivo methods to protect a subject fromdiabetes or obesity. Such methods can involve administering an AAVvirion of the embodiments to a cell, tissue, or organ outside the bodyof the subject, and then placing that cell, tissue, or organ into thebody. Such methods are known to those skilled in the art.

The dose of compositions disclosed herein to be administered to asubject to be effective (i.e., to protect a subject from diabetes orobesity) will depend on the subject's condition, manner ofadministration, and judgment of the prescribing physician. Often asingle dose can be sufficient; however, the dose can be repeated ifdesirable. In general, the dose can range from about 10⁸ virionparticles per kilogram to about 10¹² virion particles per kilogram.

The disclosure provides a treatment for diabetes. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes anexendin-4 protein. Administration of such a treatment to a subjectprotects the subject from diabetes.

The disclosure provides a treatment for obesity. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes anexendin-4 protein. Administration of such a treatment to a subjectprotects the subject from obesity.

The disclosure also provides a preventative for diabetes. Such apreventative comprises an AAV virion comprising an AAV vector thatencodes an exendin-4 protein. Administration of such a preventative to asubject protects the subject from diabetes.

The disclosure also provides a preventative for obesity. Such apreventative comprises an AAV virion comprising an AAV vector thatencodes an exendin-4 protein. Administration of such a preventative to asubject protects the subject from obesity.

The disclosure provides a salivary gland cell transfected with an AAVvector that encodes an exendin-4 protein. The salivary gland cell can bethat of a subject that is diabetic or susceptible to diabetes. Thesalivary gland cell can be that of a subject that obese or susceptibleto obesity. In one embodiment, the salivary gland cell is that of adiabetic subject. In one embodiment, the salivary gland cell is that ofan obese subject.

The disclosure provides an AAV virion comprising an AAV vector thatencodes an exendin-4 protein for the protection of a subject fromdiabetes or obesity. The disclosure provides an AAV virion comprising anAAV vector that encodes an exendin-4 protein for the protection of asubject from diabetes. The disclosure provides an AAV virion comprisingan AAV vector that encodes an exendin-4 protein for the protection of asubject from obesity. The disclosure provides an AAV virion comprisingan AAV vector that encodes an exendin-4 protein for the prevention ofsymptoms of diabetes in a subject. The disclosure provides an AAV virioncomprising an AAV vector that encodes an exendin-4 protein for thetreatment of symptoms of diabetes in a subject. The disclosure providesan AAV virion comprising an AAV vector that encodes an exendin-4 proteinfor the amelioration of symptoms of diabetes in a subject. Thedisclosure provides an AAV virion comprising an AAV vector that encodesan exendin-4 protein for the prevention of symptoms of obesity in asubject. The disclosure provides an AAV virion comprising an AAV vectorthat encodes an exendin-4 protein for the treatment of symptoms ofobesity in a subject. The disclosure provides an AAV virion comprisingan AAV vector that encodes an exendin-4 protein for the amelioration ofsymptoms of obesity in a subject

The disclosure provides for the use of an AAV virion comprising an AAVvector that encodes an exendin-4 protein for the manufacture of amedicament to protect a subject from diabetes or obesity. The disclosureprovides for the use of an AAV virion comprising an AAV vector thatencodes an exendin-4 protein for the manufacture of a medicament toprotect a subject from diabetes. The disclosure provides for the use ofan AAV virion comprising an AAV vector that encodes an exendin-4 proteinfor the manufacture of a medicament to protect a subject from obesity.The disclosure provides for the use of an AAV virion comprising an AAVvector that encodes an exendin-4 protein for the manufacture of amedicament to prevent symptoms of diabetes in a subject. The disclosureprovides for the use of an AAV virion comprising an AAV vector thatencodes an exendin-4 protein for the manufacture of a medicament totreat symptoms of diabetes in a subject. The disclosure provides for theuse of an AAV virion comprising an AAV vector that encodes an exendin-4protein for the manufacture of a medicament to ameliorate symptoms ofdiabetes in a subject. The disclosure provides for the use of an AAVvirion comprising an AAV vector that encodes an exendin-4 protein forthe manufacture of a medicament to prevent symptoms of obesity in asubject. The disclosure provides for the use of an AAV virion comprisingan AAV vector that encodes an exendin-4 protein for the manufacture of amedicament to treat symptoms of obesity in a subject. The disclosureprovides for the use of an AAV virion comprising an AAV vector thatencodes an exendin-4 protein for the manufacture of a medicament toameliorate symptoms of obesity in a subject.

The disclosure provides a method to protect a subject from an incretindefect, wherein the method comprises administering to a salivary glandof a subject an AAV virion comprising an AAV vector that encodes a GLP-1analog protein, wherein administration of the virion protects thesubject from a disease due to an incretin defect. As used herein, anincretin defect is a reduction of an incretin in a subject. A diseasedue to an incretin defect is a disease caused by a reduction in anincretin in a subject. Formulations, administration routes,administration methods, and dosages are described herein.

The disclosure provides a method to protect a subject from diabetes,wherein the method comprises administering to a salivary gland of asubject an AAV virion comprising an AAV vector that encodes a GLP-1analog protein, wherein administration of the virion protects thesubject from diabetes. Formulations, administration routes,administration methods, and dosages are described herein.

The disclosure provides a method to protect a subject from obesity,wherein the method comprises administering to a salivary gland of asubject an AAV virion comprising an AAV vector that encodes a GLP-1analog protein, wherein administration of the virion protects thesubject from obesity. Formulations, administration routes,administration methods, and dosages are described herein.

The disclosure provides a treatment for an incretin defect. Such atreatment comprises an AAV virion comprising an AAV vector that encodesa GLP-1 analog protein. Administration of such a treatment to a subjectprotects the subject from the incretin defect.

The disclosure provides a treatment for diabetes. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes a GLP-1analog protein. Administration of such a treatment to a subject protectsthe subject from diabetes.

The disclosure provides a treatment for obesity. Such a treatmentcomprises an AAV virion comprising an AAV vector that encodes a GLP-1analog protein. Administration of such a treatment to a subject protectsthe subject from obesity.

The disclosure also provides a preventative for an incretin defect. Sucha preventative comprises an AAV virion comprising an AAV vector thatencodes a GLP-1 analog protein. Administration of such a preventative toa subject protects the subject from the incretin defect.

The disclosure also provides a preventative for diabetes. Such apreventative comprises an AAV virion comprising an AAV vector thatencodes a GLP-1 analog protein. Administration of such a preventative toa subject protects the subject from diabetes.

The disclosure also provides a preventative for obesity. Such apreventative comprises an AAV virion comprising an AAV vector thatencodes a GLP-1 analog protein. Administration of such a preventative toa subject protects the subject from obesity.

The disclosure provides a salivary gland cell transfected with an AAVvector that encodes a GLP-1 analog protein. The salivary gland cell canbe that of a subject that has or is susceptible to an incretin defect.The salivary gland cell can be that of a subject that is diabetic orsusceptible to diabetes. The salivary gland cell can be that of asubject that is obese or susceptible to obesity. In one embodiment, thesalivary gland cell is that of a diabetic subject. In one embodiment,the salivary gland cell is that of an obese subject.

The disclosure provides an AAV virion comprising an AAV vector thatencodes a GLP-1 analog protein for the protection of a subject from anincretin defect. The disclosure provides for the use of an AAV virioncomprising an AAV vector that encodes a GLP-1 analog protein for themanufacture of a medicament to protect a subject from an incretindefect.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. Efforts have also beenmade to ensure accuracy with respect to nucleic acid sequences and aminoacid sequences presented, but some experimental errors and deviationsshould be accounted for. Unless indicated otherwise, parts are parts byweight, molecular weight is weight average molecular weight, andtemperature is in degrees Celsius. Standard abbreviations are used.

Example 1. Materials and Methods

In Vitro Secretion and Furin Cleavage Assays

AAV virions of the embodiments, e.g., AAV5-NGF-Ex4, were tested in vitrofor secretion of exendin-4 secretion in the cell media. 293T cells weremaintained in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum (FBS). The media contained 2 mM L-glutamine, 100 U ofpenicillin/ml, and 0.1 mg of streptomycin/ml. Cells, maintained at 37°C. under a 5% CO₂ humidified atmosphere were incubated with AAV virionAAV5-NGF-Ex4 at a multiplicity of infection of 10³ DNAse resistantparticles (DRP)/ml per cell. Furin sensitivity of the fusion proteincomprising a mouse NGF secretory segment joined to an exendin-4 proteindomain (NGF-Ex4 fusion protein) was tested by transducing 293 cellstransfected with a furin-expressing plasmid (gift of Dr Jian Cao, StonyBrook University, N.Y.). After incubation (96 hours), supernatant mediumwas tested for exendin-4 biological activity on a Chinese hamster ovarycell line stably transfected with rat GLP-1 receptor (CHO-GLP1R)accordingly to a previously reported study (Doyle et al., 2001,Endocrinology 142, 4462-4468).

Study in a Normal Animal Model

In accordance with an animal protocol approved by the Animal Care andUse Committee of the NIH/NIDCR, Balb/cJ mice (n=4) and Wistar rats (n=2)received 50 μl of 10¹¹ and 5×10″ DRP/ml of AAV virion AAV5-NGF-Ex4,respectively. At the end of the experiment, 6 weeks later, blood wascollected and serum tested for exendin-4 biological activity onCHO-GLP1R cells.

Experimental Animals and Studies Related Thereto

Additional studies were carried out in accordance with the EuropeanCommunities Council Directive of 24 Nov. 1986 (86/609/EEC) forexperimental animal care. The study protocol approved by the ItalianNational Health Institute Committee on Animal experiments. All surgerieswere performed under anesthesia, and all efforts were made to minimizesuffering. Male four-week old male CD1 mice (n=20) were purchased fromHarlan Laboratories (Udine, Italy), housed at five animals per group,and fed high fat diet (HFD) ad libitum (Dottori Piccioni LaboratoriesSrl, Milan, Italy). The HFD supplied 60% of energy as fat and 20% ascarbohydrate. The fatty acid composition was as follows: 42.0% saturatedfatty acids (palmitic and stearic acids), 43% monounsaturated fattyacids (oleic acid), and 15% polyunsaturated fatty acid (linoleic acidand linolenic acid). The carbohydrates present were cornstarch (45%),maltodextrin (50%), and sucrose (5%). The HFD contained 300 mgcholesterol/kg, and its energy density was 21.10 kJ/g. The HFD fed miceare recognized as an efficient and robust animal model for obesity,early prone to impaired glucose tolerance and T2DM development (Breslinet al., 2010, Lab Anim 44, 231-237).

Male Zucker fa/fa rats (n=10), 8 weeks of age, purchased from CharlesRiver Laboratories (Lecco, Italy), were housed in a single cage andreceived standard chow ad libitum (Purina Rodents Laboratory Diet).Zucker fa/fa rats are a spontaneous genetic obesity model, characterizedby a missense mutation in the leptin receptor gene (Oana et al., 2005,Metabolism 54, 995-1001).

Submandibular salivary glands of 9-week old Zucker fa/fa rats (n=5) and8-week old HFD mice (n=10) were transduced by a single percutaneousinjection of 50 μl of AAV virion AAV5-NGF-Ex4 (5×10′² DRP/ml). Controlanimals (n=5 rats; n=10 mice) received 50 μl of an AAV-5 CMV NGF viriondevoid of an exendin-4 transgene (empty virion).

Weight, food, water intake, urine volume, and glycemia were monitoredevery 7 days throughout the study. On a monthly basis, urine and fecescollection during overnight fasting were performed and urine volume,feces weight and water intake were determined. In order to evaluateeffects of treatment on short-term food consumption, a 120-minute foodintake evaluation, after overnight fasting, was also conducted in rats.A fixed amount of standard chow was given in individual cages androdents' food intake (evaluated as the difference between the baselineamount and the residual food, including spillage) was measured every 15minutes.

An intraperitoneal insulin tolerance test (ITT) was performed in HFDmice, 41 days following AAV virion AAV5-NGF-Ex4 administration. Eachanimal was fasted for 4 hours. Following intraperitoneal insulin(Humulin R Regular, Lilly) injection (1 UI/kg), blood samples from thelateral tail vein were collected to measure glycemia at 0, 15, 30, 60,90 and 120 minutes.

Blood samples were withdrawn at week 6 in HFD mice in order to detectEx-4, glycaemia, HbA1c, leptin and adiponectin circulating levels and at0, 4, and 8 week in Zucker fa/fa rats in order to detect Ex-4 andglucose values. HbA1c was determined at baseline and 8 weeks aftervector administration. Blood samples were obtained through jugularsampling conducted in isoflorane-anesthetized animals. At days 60 and42, rats and mice were respectively euthanized by CO₂ (80%) inhalation.Salivary gland, liver, spleen, and pancreas tissues were collected forDNA extraction and immunohistochemical staining.

Exendin-4 Assay

Circulating exendin-4 levels were determined in animal serum samplesusing a specific Enzyme Immunoassay (EIA) kit (Phoenix Europe GmbH,Germany) unable to detect endogenous GLP-1 (exendin-4 exhibits 53%structural homology to native GLP-1), according to the manufacturers'instructions. Minimum detectable concentration was 2.6 pmol/L.

Exendin-4 Biological Activity Assay

CHO/GLP-1R cells grown to 60-70% confluence on 12-well plates werewashed three times with Krebs-Ringer phosphate buffer (KRP) andincubated with 1 ml KRP containing 0.1% BSA for 2 hours at 37° C. in ahumidified air incubator. Cells were then incubated in 1 ml KRPsupplemented with 0.1% BSA with isobutylmethylxanthine (IBMX, 1 mM) inthe presence or absence of serum samples. The reaction was stopped 30minutes later by washing the intact cells three times with ice-coldphosphate-buffered saline. The intracellular cAMP was extracted byincubating the cells in ice-cold perchloric acid (0.6 M, 1 ml, 5minutes). After adjusting the pH of the samples to pH 7 using potassiumcarbonate (5 M, 84 μl), sample tubes were vortexed, and the precipitatewas sedimented by centrifugation (5 min, 2000×g, 4° C.). The supernatantwas vacuum-dried and solubilized in 0.05 M Tris (pH 7.5) containing 4 mmEDTA (300 μl). Sodium carbonate (0.15 μM) and zinc sulfate (0.15 μM)were added to the samples, which were then incubated for 15 minutes onice. The resulting salt precipitate was removed by centrifugation (5minutes, 2000×g, 4° C.). The samples were assayed in duplicate aliquots(50 μl) using a [3H]cAMP competitive binding assay kit (AmershamPharmacia Biotech, Little Chalfont, UK).

AAV Virion Biodistribution

In order to assess virion biodistribution at the end point of the study,a DNA isolation kit was used to purify total genomic DNA from salivaryglands, liver, spleen and pancreas (Wizard DNA purification kit, PromegaCorporation, Madison, Wis., USA). Quantitative PCR amplification (20 μlfinal volume) of genomic DNA (100 ng) was performed with an ABI PRISM7700 Sequence Detection System (Applied Biosystems, Foster City, Calif.)by using the SYBR Green PCR Master Mix and a specific 5′ and 3′ primerpair appropriate (0.3 μM; CMV forward5′-CATCTACGTATTAGTCATCGCTATTACCAT-3′, CMV reverse5′-TGGAAATCCCCGTGAGTCA-3′) for CMV promoter. Amplification and detectionwere performed with an ABI Prism 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.). A PCR cycling reaction involved aninitial hold at 95° for 10 minutes, followed by cycling conditions of95° C. for 15 seconds, 60° C. for 1 min for 40 cycles. The viral DNA ineach sample was quantified by comparing the fluorescence amplificationprofiles with a set of DNA standards using AAV5 virion and 100 ng ofgenomic DNA of untreated animals for each specific tissue. Eachmeasurement was carried out in duplicate. Data are expressed in copiesof AAV5 for 100 ng of genomic DNA.

Salivary Glands Immunohistochemical Assay

At the end of the study, salivary glands were removed from treated (n=5)and control (n=5) HFD mice, fixed in 4% paraformaldehyde for 24 hours atroom temperature, cryoprotected in 30% sucrose in phosphate-bufferedsaline (PBS) for approximately 12 hours at 4° C. and then embedded inKillik cryostat embedding medium (Bio-Optica, Milan Italy).Cryosections, 10-μm thick, were collected on polylysine-coated slides.The slides were pre-incubated in 0.5% Triton (Sigma Aldrich, Milan,Italy) and 1.5% bovine serum albumin (BSA) (Sigma Aldrich, Milan, Italy)in PBS for 15 minutes at room temperature to saturate non-specificsites. The sections then were incubated 24 hours at 4° C. with a primaryantibody against exendin-4 (Phoenix Europe GmbH, Germany) at a finaldilution of 1:50. Subsequently, the sections were incubated with anAlexa Fluor 488 secondary donkey anti-rabbit antibody (Invitrogen, SanDiego, Calif., USA) at a final dilution of 1:333 for 2 hours at roomtemperature. The immunoreaction products were observed under anepifluorescence Zeiss Axioskop microscope (Zeiss, Germany) at ×40magnification.

Adipokines Circulating Levels Assay

Serum leptin and adiponectin levels were assayed only in the polygenicmodel of obesity and T2DM HFD mice using a commercially available kitaccording to manufacturer's instructions. A sandwich enzyme immunoassay(ELISA) was used for the quantitative measurement of mouse proteins(Biovendor, Heidelberg, Germany and B-Bridge International Inc., CA,USA, for leptin and adiponectin respectively). Intra- and inter-assaycoefficients of variation were less than 5%.

Visceral Adipose Tissue Adipokines Profile: RNA Extraction and Real TimePCR Determinations

Total RNA was extracted from 50 mg of mice visceral adipose tissue.Briefly, tissue samples were collected, immediately snap frozen inliquid nitrogen and disrupted by homogenization in QIAzol Lysis Reagentusing the TissueLyser (QIAGEN GmbH, Hilden, Germany). RNA was extractedusing RNeasy Lipid Tissue Mini Kit (QIAGEN GmbH, Hilden, Germany)according to the manufacturer's instructions. One μg of RNA was treatedwith TURBO DNA-Free™ DNase Kit (Ambion, Inc., Austin, Tex., USA) andreverse-transcribed into cDNA for 1 h at 37° C. in a 50 μl reactioncontaining 1×RT buffer, 150 ng random hexamers, 0.5 mmol/l dNTPs, 20units of RNAsin Ribonuclease Inhibitor (Promega Corporation, Madison,Wis., USA) and 200 units of M-MLV RT (Promega Corporation, Madison,Wis., USA).

Real Time quantitative PCR was carried out on DNA Engine Opticon™ 2Continuous Fluorescence Detection System (MJ Research, MA, USA), usingPlatinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen Corporation, CA,USA) and 300 nM specific primers for each gene: 18s forward 5′-CGG CTACCA CAT CCA AGG AA-3′, reverse 5′-GCT GGA ATT ACC GCG GCT-3′; leptinforward: 5′-TCC AGA AAG TCC AGG ATG ACA C-3′, reverse: 5′-CAC ATT TTGGGA AGG CAG G-3′; adiponectin forward: 5′-ACA ATG GCA CAC CAG GCC GTGA-3′, reverse: AGC GGC TTC TCC AGG CTC TCC TTT-3′. Each cDNA sample wasassayed in duplicate and a no-template control was included in everyreaction. For each sample, gene expression values were normalized by 18sRNA content and reported as AU ratio.

Blood and Urine Analysis

Glycemic values were determined in the morning, after overnight fasting.Blood was obtained via tail vein and tested, using an Accu-Chek AvivaNano meter (Roche). HbA1c percentage values were measured on 5 μl ofwhole blood using an A1CNow+test Kit (Bayer). Urine analysis wasperformed by a colorimetric method (AUTION Sticks 10TA; Arkray, Inc.,Kyoto, Japan) in order to detect glucose, protein, bilirubin,urobilinogen, pH, specific gravity, blood, ketone, nitrite and leukocytelevels.

Statistical Analysis

The statistical significance of differences between experimental andcontrol groups was analyzed by Student's t-test. p<0.05 was consideredstatistically significant. Values are presented as mean±standard error(SE).

Example 2. Production of AAV Vectors and AAV Virions Encoding anExendin-4 Protein of the Embodiments

A nucleic acid molecule encoding an exendin-4 protein having amino acidsequence SEQ ID NO:8 joined to a secretory segment from murine nervegrowth factor (NGF), which was modified to be cleaved by a furinprotease, in order to facilitate processing and secretion of theexendin-4 protein was produced. The NGF secretory segment was found tobe more efficient than other secretory segments, e.g., the Factor IXsecretory segment (data not shown). The encoded fusion protein, referredto herein as NGF-Ex4, is represented by amino acid sequence SEQ ID NO:2.

The AAV5-NGF-Ex4 expression cassette in the plasmid vectorpAAV-CMV-NGF-exendin-4, also referred to as pAAV5-NGF-Ex4, was designedto contain the cytomegalovirus (CMV) promoter, the mouse nerve growthfactor (NGF) signal peptide, which had been shown to mediate secretoryexpression of polypeptides in vitro and in vivo (Beutler et al., 1995, JNeurochem 64, 475-481; Finegold et al., 1999, Hum Gene Ther 10,1251-1257) and the sequence encoding Gila monster (Heloderma suspectum)exendin-4.

Recombinant AAV virions, referred to herein as AAV5-CMV-NGF-Ex4 orAAV5-NGF-Ex4, were produced using a four-plasmid procedure as previouslydescribed (di Pasquale et al., ibid.). Briefly, semi-confluent humanembryonic kidney 293T cells, obtained from the American Type CultureCollection (ATCC, Manassas, Va.) were transfected by calcium phosphatewith four plasmids: an adenovirus helper plasmid (pAd12) containing VARNA and coding for the E2 and E4 proteins; two AAV helper plasmidscontaining either the AAV2rep or the AAV5 capsid gene and a vectorplasmid including the AAV inverted terminal repeats flanking theexendin-4 expression cassette (e.g., pAAV5-NGF-Ex4, depicted in FIG. 8).The cells were harvested 48 hours post-transfection and a crude virallysate was obtained after three freeze-thaw cycles. The clarified lysate(obtained by further low-speed centrifugation) was treated with 0.5%deoxycolic acid (DOC) and 100 U/ml DNase (Benzonase) for 30 minutes at37° C. The AAV virions were purified using CsCl gradients. The number ofAAV genomes was estimated using quantitative real-time PCR (qPCR)(Applied Biosystems, Foster City, Calif.). Immediately beforeexperiments, the AAV virions were dialyzed against 0.9% NaCl.

Example 3. Exendin-4 Expression and Secretion In Vitro

This Example indicates that fusion protein NGF-Ex4 could be secretedfrom cells transfected with AAV virion AAV5-NGF-Ex4 in culture.

Media collected from 293T cells transduced with AAV virion AAV5-NGF-Ex4produced an average of 38.3±10.4 pmol/L when assayed for exendin-4biological activity on a Chinese hamster ovary cell line stablytransfected with rat GLP-1 receptor (CHO/GLP-1R). Many cells naturallyproduce the furin protease; however the overexpression of furin, bytransfection of a plasmid encoding the protease, increased the activeexendin-4 in the medium to 75.6±11.0 pmol/L.

Example 4. Exendin-4 Expression and Secretion In Vivo

This Example demonstrates that fusion protein NGF-Ex4 is secreted frommice and rats administered a single dose of AAV virion AAV5-NGF-Ex4 totheir salivary glands.

The salivary glands of Balb/cJ mice (n=4) and Wistar rats (n=2) wereadministered a single dose of AAV virion AAV5-NSF-Ex4 at 1×10¹¹ and5×10″ DNAse resistant particles (DRP)/ml respectively. After 6 weeks,sera were tested for the presence of biologically active exendin-4.Circulating levels of exendin-4 were detected at 70.2±9.1 pmol/L and144.2±9.8 pmol/L in mice and rats, respectively.

Expression was also tested in two different models of obesity and T2DM:High Fat-Diet (HFD) mice and Zucker fa/fa rats. These animals eachreceived a single dose of 5×10¹² DRP/ml of AAV virion AAV5-NGF-Ex intotheir salivary glands. In AAV5-NGF-Ex4 treated mice (n=10), serumexendin-4 levels averaged 138.9±42.3 pmol/L at day 42, when assayed by aspecific Enzyme Immunoassay (EIA) kit, as shown in FIG. 1. InAAV5-NGF-Ex4 treated Zucker fa/fa rats (n=5), the mean circulatingexendin-4 level was 238.2±72 pmol/L at day 30 and increased to 3.25nmol/L at day 60, as shown in FIG. 1. In control animals, averagecirculating exendin-4 levels were less than 2.6 pol/L, thus below thelimit of detection, at week 6 in mice and at both week 4 and week 8 inrats. These data indicate that, surprisingly, exendin-4 produced by thesalivary glands can traffic through the cell via the endocrine pathway,resulting in circulating serum levels. The biological activity ofexendin-4 was also confirmed on CHO/GLP-1R cells (data not shown).

Expression was also confirmed by immunohistochemical staining forexendin-4 in salivary gland tissue sections from AAV5-NGF-Ex4 treated(n=5) and control (n=5) HFD mice euthanized at day 42. FIG. 2demonstrates that exendin-4 expression was observed only in theAAV5-NGF-Ex4 treated group. Only salivary ductal cells revealed positivestaining, which is consistent with the tissue tropism of AAV5 virions.

Example 5. Biodistribution of AAV Virion AAV5-NGF-Ex4

Previous studies have suggested that the vast majority of AAV virionsdelivered to the salivary glands remain in the gland. In order to assessAAV5-NGF-Ex4 biodistribution, DNA samples were collected from thesalivary glands, liver, spleen and pancreas of HFD mice (n=5 forAAV5-NGF-Ex4 treated mice and n=5 for naive mice) at the end of thestudy, and virion copy number was determined by quantitative polymerasechain reaction (qPCR) amplification using specific primers for the CMVpromoter contained in the AAV virion. Naive animals yielded backgroundlevels of 55±29 copies per 100 ng of DNA extracted from salivary glands.In AAV5 NGF-Ex4 treated HFD mice, a 60-fold increase in virion copynumber was detected in salivary glands (3551±1618 copies per 100 ng ofDNA). The virion copy number detected in other tissues such as liver(154±56 copies per 100 ng of DNA), spleen (65±23 copies per 100 ng ofextracted DNA) and pancreas (104±47 copies per 100 ng of DNA) were at ornear background levels.

Example 6. Glycemic and Extra-Glycemic Effects of Salivary GlandAdministration of AAV Virion AAV5-NGF-Ex4

To assess the glycemic and extra-glycemic effects of exendin-4expression, weight gain as well as blood and urine chemistry weremonitored throughout the study. At baseline (8 weeks of age), micetreated with salivary gland administration of AAV virion AAV5-NGF-Ex4(n=10) were not significantly different from control animals (n=10) withrespect to weight, fasting glucose, HbA1c, glycosuria, or daily foodintake, as shown in Table 1. AAV5-NGF-Ex4 treated and control micereceived the High Fat Diet ad libitum in order to develop an obesityphenotype and continued to gain weight throughout the study (FIG. 3A).However, at the termination of the study (day 42), AAV5-NGF-Ex4 treatedHFD mice had a significantly lower weight gain in comparison to controlanimals, as also shown in FIG. 3A.

TABLE 1 Baseline characteristics of High Fat Diet (HFD) fed mice andZucker fa/fa rats (control and AAV5-NGF-Ex-4 treated animals) AAV5 AAV5Control Ex-4 Ex-4 HFD HFD Control Zucker mice mice P* Zucker fa/fa ratsfa/fa rats P* n 10 10 5 5 Weight (g) 23.3 ± 1.9   23 ± 1.6 p > 0.05290.6 ± 26.2  294 ± 28.5 p > 0.05 Fasting glycaemia 4.6 ± 0.8 4.7 ± 0.6p > 0.05  5.1 ± 0.8 5.3 ± 0.8 p > 0.05 (mmol/L) HbA1c (%) <4 <4 p > 0.05 4.2 ± 0.1 4.1 ± 0.2 p > 0.05 Glycosuria  0  0 p > 0.05 0 0 p > 0.05(number positive) Daily food intake (g) 2.9 ± 0.8 3.1 ± 0.5 p > 0.0527.8 ± 2.8  29 ± 3.0 p > 0.05 *Control in comparison to AAV5 Ex-4 HFDmice and control versus AAV5 Ex-4 Zucker fa/fa rats, respectively.

Zucker fa/fa rats are a spontaneous monogenic model of obesity, as aresult of a dysfunctional leptin receptor. At baseline (9 weeks of age),Zucker fa/fa rats treated with AAV5-NGF-Ex4 virion (n=5) presented nosignificant difference in comparison to control animals (n=5) regardingweight, fasting glucose, HbA1c levels, glycosuria, and daily foodintake, as shown in Table 1. These spontaneous monogenic obesity ratsreceived standard chow ad libitum and continued to gain weightthroughout the study (FIG. 3B), which was terminated 60 days after AAVvirion AAV5-NGF-Ex4 administration. However by day 35, AAV5-NGF-Ex4treated Zucker fa/fa rats had a statistically significant reduction inweight gain compared to control animals, which persisted for theduration of the study, as shown in FIG. 3B.

In addition to reduced weight gain, AAV5-NGF-Ex4 treated HFD mice hadsignificantly lower leptin circulating levels at day 42 in comparison tocontrol animals (2.24±0.39 versus 5.89±1.07 ng/ml; p<0.01). In contrast,no significant difference in serum adiponectin levels (9.75±0.69 versus10.57±0.97 mg/l; P=NS) was observed. The reduction in leptin circulatinglevels correlated with a significant reduction in visceral adiposetissue leptin mRNA expression compared to that in control animals(3.43±0.48 versus 8.28±0.72 Arbitrary Unit, AU; p<0.01). No differencein visceral adipose tissue adiponectin mRNA expression (8.28±0.72 versus8.95±1.8 AU; P=NS) was detected.

Mice fed a high fat diet develop T2DM after 12 weeks. In order to betterunderstand early effects of AAV virion AAV5-NGF-Ex-4 treatment on thedevelopment of insulin resistance, insulin tolerance was tested (ITT)following an intraperitoneal insulin injection. FIG. 4 demonstrates thatAAV virion AAV5-NGF-Ex4 treated HFD mice, at week 6, exhibited a greaterinsulin-induced reduction in glycemia 15, 30 and 60 minutes following anintraperitoneal insulin tolerance test compared with control HFD mice.Glycemic AUC values were also significantly different betweenAAV5-NGF-Ex4 treated and control HFD mice (p<0.05). No significantdifference was observed in fasting glycemia, glycosuria or HbA1c valuesthroughout the study, as shown in Table 2.

TABLE 2 Final characteristics of High Fat-Diet (HFD) fed mice and Zuckerfa/fa rats (control and AAV5 Ex-4 treated animals). AAV5 AAV5 ControlEx-4 Ex-4 HFD HFD Control Zucker mice mice P* Zucker fa/fa rats fa/farats P* n 10 10 5 5 Weight gain (g) 19.5 ± 1.9  16.5 ± 2.7  P < 0.01241.4 ± 22.5  222 ± 23.4 P < 0.05 Fasting glycaemia 4.9 ± 0.9 4.8 ± 0.7p > 0.05  5.7 ± 0.4 5.6 ± 0.5 p > 0.05 (mmol/L) HbA1c (%) 4.2 ± 0.2 4.1± 0.2 p > 0.05  5.0 ± 0.1 4.7 ± 0.2 P < 0.05 Glycosuria  0  0 p > 0.05 40 p < 0.05 (number positive) Daily food intake (g) 4.3 ± 0.3 4.6 ± 0.3p > 0.05 21.2 ± 2.1 21.3 ± 1.9  p > 0.05 *Control in comparison to AAV5Ex-4 HFD mice and control versus AAV5 Ex-4 Zucker fa/fa rats,respectively.

In contrast, as shown in Table 2, AAV virion AAV5-NGF-Ex4 treatment ofZucker fa/fa rats resulted in significantly lower HbA1c levels ascompared with control mice (4.7±0.1 versus 5.0±0.1%; p<0.05). In controlanimals, glycosuria (>2.78 mmol/L) was detected in 4 and 2 Zucker fa/farats 30 and 60 days after virion administration, respectively. Noglycosuria was reported in AAV virion AAV5-NGF-Ex4 treated ratsthroughout the study. Accordingly, with respect to the low hypoglycemicrisk profile of exendin-4, no significant difference in fasting glycemiawas observed between treated and control rats during the study (5.7±0.40versus 5.6±0.55 mmol/L; p>0.05).

Example 7. Effect of AAV Virion AAV5-NGF-Ex4 on Food Consumption

Food consumption was also monitored throughout the study. As shown inFIG. 5, differences in daily food intact between AV5-NGF-Ex4 treated andcontrol HFD animals were detected only transiently in rats (FIG. 5B) butnot in mice (FIG. 5A). Similarly, monitoring of short-term food intakeindicated a reduction in food consumption by AAV5-NGF-Ex4 treated Zuckerfa/fa rats compared to control animals by 75 minutes (FIG. 5C).

Example 8. Conclusions and Discussion

The studies reported herein have characterized the safety profile aswell as metabolic effects, e.g., glycemic, and extra-glycemic effects,of exendin-4 expressed continuously by salivary glands of high fat-diet(HFD) mice and Zucker fa/fa rats, following AAV virion AAV5-NGF-Ex4mediated transduction of the salivary glands. The study alsocharacterized the site-specific secretion profile of sustained exendin-4expression by the salivary glands of both the mouse and rat models.Exendin-4 produced by the salivary gland was well tolerated, andresulted in a significant decrease in weight gain, improved glucosehomeostasis and an improved visceral adipose tissue adipokine profile inthese two different animal models of obesity and T2DM, suggestinglong-term benefit following sustained expression.

More specifically, exendin-4 is a glucagon-like peptide 1 (GLP-1)receptor agonist approved for the treatment of Type 2 Diabetes (T2DM),which requires twice-daily subcutaneous administration. The aim of thesestudies was to characterize the site-specific profile and metaboliceffects (e.g., glycemic and extra-glycemic effects) of exendin-4 levelsexpressed continuously from the salivary glands in vivo, followingadeno-associated virus-mediated (AAV) gene therapy. Following a directinjection into the salivary glands of two different rodent models ofobesity/T2DM, specifically Zucker fa/fa rats and high fed diet (HFD)mice, biologically active exendin-4 was detected in the blood of bothanimal models and expression persisted in salivary gland ductal cellsuntil the end of the study. In treated mice, Ex-4 levels averaged138.9±42.3 pmol/L on week 6 and in treated rats, mean circulating Ex-4level were 238.2±72 pmol/L on week 4 and continued to increase throughweek 8. AAV virion expression was only detected in salivary gland tissueand localized to ductal cells within the gland. Expression of exendin-4from AAV virion AAV-5-NGF-Ex4 resulted in significantly decreased weightgain as well as in glucose homeostasis improvement in both mice fed ahigh fat diet and in Zucker fa/fa rats. Mice also exhibited asignificant adipokine profile improvement and lower expression of leptinin visceral adipose tissue. These findings indicate that sustained,site-specific, expression of exendin-4 following AAV-mediated genetherapy is well tolerated and has utility in the treatment of bothmonogenic- and polygenic forms of obesity and/or T2DM.

GLP-1 receptor agonists are a very interesting therapeutic approach forthe treatment of T2DM, showing a remarkable efficacy on glycemic control(e.g., blood glucose and HbA1c), with low hypoglycemic risk andbeneficial effects on body weight and other cardiovascular risk factors,such as lipid profile and blood pressure (see, for example, Rotella etal., 2005, J Endocrinol Invest 28, 746-758; Monami et al., 2009, Eur JEndocrinol 160, 909-917). Furthermore, phase II clinical trials haveshown the potential efficacy and safety of GLP-1 receptor agonists inthe treatment of obesity (Astrup et al., 2009, Lancet 374, 1606-1616),although this disease is not among the approved indication for theseagents in any country. Wider use of GLP-1 receptor agonists is presentlylimited by their cost, and need for multiple subcutaneousadministration, e.g., twice daily, which is not accepted by somepatients.

Plasmid DNA and adenoviral mediated gene therapy can direct theexpression of GLP-1 receptor agonists in tissues not physiologicallyintended for secretion (see, for example, Voutetakis et al., 2010,Endocrinology 151, 4566-4572; Kumar et al., 2007, Gene Ther 14, 162-172;Liu et al., 2010, Biochem Biophys Res Commun 403, 172-177; Samson etal., 2008, Mol Ther 16, 1805-1812 (erratum in Mol Ther 17, 1831); Lee etal., 2008, J Gene Med 10, 260-268; Choi et al., 2005, Mol Ther 12,885-891; Lee et al., 2007, Diabetes 56, 1671-1679), achieving long-termmetabolic effects through high vector doses administered systemically,either by intravenous or intraperitoneal injection. Both systemsdemonstrated short-term efficacy of metabolic improvement and requiredhigh vector doses and/or systemic administration. Recently, Voutetakiset al. reported that an adenoviral-mediated transduction of salivaryglands with a vector encoding GLP-1 can induce short-term moderatereduction of blood glucose in a murine model of diabetes (Voutetakis etal, 20120, Endocrinology 151, 4566-4572). Not surprisingly, althoughthose approaches were shown to reduce blood glucose, no effect on HbA1clevels has ever been reported, confirming that the therapeutic efficacywas not sustained.

The use of exendin-4 instead of GLP-1 has several advantages as a resultof its much longer half-life. The studies reported herein show, for thefirst time, sustained secretion of exendin-4 at pharmacological levelsfrom salivary glands. These circulating levels are several-fold higherthan reported for endogenous human GLP-1 after a meal (40 pmol/l; Orskovet al., 1994, Diabetes 43, 535-539), and exendin-4 steady-state valuesattained in human clinical studies with 10 μg injected exenatide (50pmol/l; Kim et al., 2007, Diabetes Care 30, 1487-1493). This sustained,AAV5 mediated, exendin-4 expression and secretion resulted in asignificant reduction in weight gain, which persisted for the durationof the study. The mechanism underlying the effect of exendin-4 on bodyweight is still controversial, and is likely related to a peripheralaction on gastric motility and/or a direct effect on the hypothalamicregion involved in the regulation of eating behavior. Effects on dailyfood intake between treated and control animals were detected onlytransiently in Zucker fa/fa rats, which showed a reduced meal size,suggesting enhancement of satiety. It should be noted that limitationson the accuracy of measuring food intake could have prevented detectionof a difference in food intake associated with changes in body weightover the long-term. The weight loss could also explain the enhancedinsulin sensitivity observed in AAV5-NGF-Ex4 treated HFD mice, however adirect insulin-sensitizing effect of exendin-4 is also possible.Alternatively, the improvement in insulin sensitivity could be due tothe inhibition of glucagon secretion. Metabolic effects of AAV5-Ex4mediated gene therapy included a significant reduction in HbA1c levelsand glycosuria in treated versus control Zucker fa/fa rats. Although theeffect of exendin-4 expression could have contributed to the improvementin glycemic control, it is very likely that direct actions of exendin-4on insulin and glucagon secretion, and possibly insulin resistance,played a major role.

This study indicates that an alternative approach to deliveringexendin-4 is possible and can reduce weight gain as well as triggerimproved metabolic function in two animal models. Although exendin-4 hasshown metabolic benefits, there are concerns about the long-term safetyof this drug and its effect on inducing tumors in rodents (Knudsen etal., 2010, Endocrinology 151:1473-1486), which has not been reported inhumans. This affect may be the result of the high bolus delivery ofexendin-4 necessary when the drug is given by injection and would not beexpected with gene therapy-based delivery, which has been shown forother systems to be able to maintain a constant level of expression.

Other studies have demonstrated that transgene expression in rodents canlast of the life of the animal following gene transfer to severaltissues including salivary glands (Voutetakis et al, 2005, ibid.).Although, no adverse effects of sustained expression were noted ineither animal model over the 2-month period of this study, additionallong-term studies would support the long-term safety of AAV-mediatedexendin-4 gene transfer.

Type 2 diabetes and obesity are growing public health problem worldwide,deserving the definition of “epidemic” (see, for example, the WorldHealth Organization Global InfoBase). There is a paucity of effectivedrugs to treat obesity; therefore time-consuming and expensivenon-pharmacological approaches are the only ones that can be used inpatients for whom bariatric surgery is not indicated. On the other hand,the management of T2DM is centered on multiple pharmacotherapies, withan increasing burden on a patient's quality of life. This studyindicates that alternative approaches are possible, deliveringtherapeutics agents in a safe and effective way, which does not requireregular administration of a drug. AAV5-mediated transgene expression ofexendin-4 in salivary glands determines a sustained reduction of bodyweight, blood glucose and HbA1c in different animal models of obesityand diabetes, with no relevant side effects, and without the involvementof any organ critical for life.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims.

What is claimed is:
 1. A nucleic acid molecule encoding a fusion proteincomprising an exendin-4 protein joined to a secretory segment; whereinthe fusion protein: (i) is encoded by a polynucleotide sequence at least80% identical to SEQ ID NO:5, wherein the encoded fusion protein effectsan agonist response at a GLP-1 receptor, (ii) comprises an amino acidsequence at least 80% identical to SEQ ID NO:2 or SEQ ID NO:6, whereinfusion protein effects an agonist response at a GLP-1 receptor, or (iii)comprises SEQ ID NO:2 or SEQ ID NO:6.
 2. The nucleic acid molecule ofclaim 1, wherein the fusion protein is encoded by a polynucleotidesequence at least 80% identical to SEQ ID NO:5, wherein the encodedfusion protein effects an agonist response at a GLP-1 receptor.
 3. Thenucleic acid molecule of claim 1, wherein the fusion protein comprisesan amino acid sequence at least 80% identical to SEQ ID NO:2 or SEQ IDNO:6, wherein fusion protein effects an agonist response at a GLP-1receptor.
 4. The nucleic acid molecule of claim 1, wherein the fusionprotein comprises SEQ ID NO:2 or SEQ ID NO:6.
 5. An adeno-associatedvirus (AAV) vector comprising a nucleic acid molecule encoding a fusionprotein comprising an exendin-4 protein joined to a secretory segment;wherein the fusion protein: (i) is encoded by a polynucleotide sequenceat least 80% identical to SEQ ID NO:5, wherein the encoded fusionprotein effects an agonist response at a GLP-1 receptor, (ii) comprisesan amino acid sequence at least 80% identical to SEQ ID NO:2 or SEQ IDNO:6, wherein fusion protein effects an agonist response at a GLP-1receptor, or (iii) comprises SEQ ID NO:2 or SEQ ID NO:6.
 6. An AAVvirion comprising the AAV vector of claim claim
 5. 7. The AAV vector ofclaim 5, wherein the fusion protein is encoded by a polynucleotidesequence at least 80% identical to SEQ ID NO:5, wherein the encodedfusion protein effects an agonist response at a GLP-1 receptor.
 8. AnAAV virion comprising the AAV vector of claim
 7. 9. The AAV vector ofclaim 5, wherein the fusion protein comprises an amino acid sequence atleast 80% identical to SEQ ID NO:2 or SEQ ID NO:6, wherein fusionprotein effects an agonist response at a GLP-1 receptor.
 10. An AAVvirion comprising the AAV vector of claim
 9. 11. The AAV vector of claim5, wherein the fusion protein comprises SEQ ID NO:2 or SEQ ID NO:6. 12.An AAV virion comprising the AAV vector of claim 11.